Synthesis and aggregation behaviour of a new sultaine surfactant

6
Colloids and Surfaces B: Biointerfaces 87 (2011) 73–78 Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces jou rn al h om epage: www.elsevier.com/locate/colsurfb Synthesis and aggregation behaviour of a new sultaine surfactant Paolo De Maria a , Antonella Fontana a , Gabriella Siani a , Eleonora D’Aurizio a , Giorgio Cerichelli b , Marco Chiarini c , Guido Angelini a , Carla Gasbarri a,a Dipartimento di Scienze del Farmaco, Università G. d’Annunzio, via dei Vestini, 66013 Chieti, Italy b Dipartimento di Chimica, Ingegneria Chimica e Materiali, Università degli Studi de L’Aquila, via Vetoio, 67010 Coppito, L’Aquila, Italy c Dipartimento di Scienze degli Alimenti, Università degli Studi di Teramo, via Lerici, 64023 Mosciano Sant’Angelo, Teramo, Italy a r t i c l e i n f o Article history: Received 7 December 2010 Received in revised form 1 April 2011 Accepted 3 May 2011 Available online 10 May 2011 Keywords: Surfactant Vesicles CVC Kinetics Fluorescence a b s t r a c t The aggregation properties of a new sultaine surfactant have been studied in buffered aqueous solution at pH 7.4 under controlled condition of osmolarity. Spontaneously formed sultaine vesicles with a mean diameter of about 1 m can be observed by opti- cal microscopy. The phase behaviour of the surfactant has been investigated by differential scanning calorimetry (DSC) and Nile Red fluorescence. Two critical vesicular concentrations (CVC 1 and CVC 2 ) have been fluorimetrically measured, by using pyrene and Nile Red as the fluorescent probes. The two populations of vesicles behave differently as a consequence of their size. The stability of extruded large unilamellar vesicles (LUV) formed slightly above the CVC 1 has been evaluated in the temperature range 25–75 C by following the rate of spontaneous release of entrapped 5(6)-carboxyfluorescein (CF). The stability of the same vesicles at 70 C has also been investigated under osmotic stress obtained by adding NaCl or sucrose to the bulk solution. At a sultaine concentration above the CVC 2 LUV tend to associate and form stable larger closely packed aggregates as suggested by Dynamic Laser Light Scattering and rheological measurements. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Surfactants can self-assemble into a large variety of structures, different in size, shape and morphology, depending of their critical packing parameters [1]. Different structures and phase transitions can be triggered by changing the conditions under which the aggre- gation takes place. Such variations can be induced, for example, by changing the surfactant concentration, the ionic strength, the tem- perature of the solution [2], the application of external magnetic or electric fields [3] and the input of external energy (e.g. sonication [4] or extrusion [5]). Although the phase behaviour of many synthetic or natural surfactants has been widely studied, less is known about the stabil- ity (kinetics and thermodynamics) of the aggregates, despite their potential role in pharmaceutical or cosmetic formulations. Double chain surfactants generally form bilayered structures in aqueous solutions. The two alkyl chains do not need to be of the same length and the formation of vesicles may be favoured over the assembly in planar bilayers by increasing the chain length asymmetry [6,7]. Corresponding author. Tel.: +39 0871 3554786; fax: +39 0871 3554791. E-mail address: [email protected] (C. Gasbarri). Vesicles from a totally synthetic surfactant bearing two hydro- carbon tails of equal length was first reported by Kunitake and Okahata in 1977 [8]. In that work transmission electron microscopy of a sonicated aqueous solution of the cationic double chain surfac- tant didodecyldimethylammonium bromide (DDAB) highlighted the formation of vesicles with a mean diameter of 400 ˚ A. The DDAB ability to self-assemble into vesicles in diluted solu- tion [9,10] was also studied in binary [11,12] or ternary [13,14] mixtures with cosurfactants. Moreover, at 25 C in pure water [15] or in aqueous lecithin [16], collapsed and swollen lamellar phases may be distinguished at relatively high DDAB concentration. The collapsed lamellar phase corresponds to planar bilayer structures, while the swollen lamellar phase forms when densely packed vesi- cles increase their size as the water content decreases. Water-soluble zwitterionic surfactants have attracted consider- able attention especially due to their many applications [17], low irritancy and non denaturizing-protein effect. Vesicles from natural or synthetic surfactants may also act as useful carriers of phar- maceutical and cosmetic agents in biotechnological applications [18]. To be suitable as a drug delivery system, a surfactant vesicle should be stable enough under physiological conditions to release its content in a controlled way. The aim of the present work is to investigate the aggre- gation properties of a new synthetic zwitterionic surfactant, 0927-7765/$ see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.05.003

Transcript of Synthesis and aggregation behaviour of a new sultaine surfactant

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Colloids and Surfaces B: Biointerfaces 87 (2011) 73– 78

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

jou rn al h om epage: www.elsev ier .com/ locate /co lsur fb

ynthesis and aggregation behaviour of a new sultaine surfactant

aolo De Mariaa, Antonella Fontanaa, Gabriella Siania, Eleonora D’Aurizioa, Giorgio Cerichelli b,arco Chiarini c, Guido Angelinia, Carla Gasbarri a,∗

Dipartimento di Scienze del Farmaco, Università G. d’Annunzio, via dei Vestini, 66013 Chieti, ItalyDipartimento di Chimica, Ingegneria Chimica e Materiali, Università degli Studi de L’Aquila, via Vetoio, 67010 Coppito, L’Aquila, ItalyDipartimento di Scienze degli Alimenti, Università degli Studi di Teramo, via Lerici, 64023 Mosciano Sant’Angelo, Teramo, Italy

r t i c l e i n f o

rticle history:eceived 7 December 2010eceived in revised form 1 April 2011ccepted 3 May 2011vailable online 10 May 2011

eywords:urfactantesicles

a b s t r a c t

The aggregation properties of a new sultaine surfactant have been studied in buffered aqueous solutionat pH 7.4 under controlled condition of osmolarity.

Spontaneously formed sultaine vesicles with a mean diameter of about 1 �m can be observed by opti-cal microscopy. The phase behaviour of the surfactant has been investigated by differential scanningcalorimetry (DSC) and Nile Red fluorescence.

Two critical vesicular concentrations (CVC1 and CVC2) have been fluorimetrically measured, by usingpyrene and Nile Red as the fluorescent probes. The two populations of vesicles behave differently as aconsequence of their size.

VCineticsluorescence

The stability of extruded large unilamellar vesicles (LUV) formed slightly above the CVC1 has beenevaluated in the temperature range 25–75 ◦C by following the rate of spontaneous release of entrapped5(6)-carboxyfluorescein (CF). The stability of the same vesicles at 70 ◦C has also been investigated underosmotic stress obtained by adding NaCl or sucrose to the bulk solution.

At a sultaine concentration above the CVC2 LUV tend to associate and form stable larger closely packedaggregates as suggested by Dynamic Laser Light Scattering and rheological measurements.

. Introduction

Surfactants can self-assemble into a large variety of structures,ifferent in size, shape and morphology, depending of their criticalacking parameters [1]. Different structures and phase transitionsan be triggered by changing the conditions under which the aggre-ation takes place. Such variations can be induced, for example, byhanging the surfactant concentration, the ionic strength, the tem-erature of the solution [2], the application of external magnetic orlectric fields [3] and the input of external energy (e.g. sonication4] or extrusion [5]).

Although the phase behaviour of many synthetic or naturalurfactants has been widely studied, less is known about the stabil-ty (kinetics and thermodynamics) of the aggregates, despite theirotential role in pharmaceutical or cosmetic formulations.

Double chain surfactants generally form bilayered structuresn aqueous solutions. The two alkyl chains do not need to be of

he same length and the formation of vesicles may be favouredver the assembly in planar bilayers by increasing the chain lengthsymmetry [6,7].

∗ Corresponding author. Tel.: +39 0871 3554786; fax: +39 0871 3554791.E-mail address: [email protected] (C. Gasbarri).

927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2011.05.003

© 2011 Elsevier B.V. All rights reserved.

Vesicles from a totally synthetic surfactant bearing two hydro-carbon tails of equal length was first reported by Kunitake andOkahata in 1977 [8]. In that work transmission electron microscopyof a sonicated aqueous solution of the cationic double chain surfac-tant didodecyldimethylammonium bromide (DDAB) highlightedthe formation of vesicles with a mean diameter of 400 A.

The DDAB ability to self-assemble into vesicles in diluted solu-tion [9,10] was also studied in binary [11,12] or ternary [13,14]mixtures with cosurfactants. Moreover, at 25 ◦C in pure water [15]or in aqueous lecithin [16], collapsed and swollen lamellar phasesmay be distinguished at relatively high DDAB concentration. Thecollapsed lamellar phase corresponds to planar bilayer structures,while the swollen lamellar phase forms when densely packed vesi-cles increase their size as the water content decreases.

Water-soluble zwitterionic surfactants have attracted consider-able attention especially due to their many applications [17], lowirritancy and non denaturizing-protein effect. Vesicles from naturalor synthetic surfactants may also act as useful carriers of phar-maceutical and cosmetic agents in biotechnological applications[18]. To be suitable as a drug delivery system, a surfactant vesicle

should be stable enough under physiological conditions to releaseits content in a controlled way.

The aim of the present work is to investigate the aggre-gation properties of a new synthetic zwitterionic surfactant,

74 P. De Maria et al. / Colloids and Surfaces

Fig. 1. Structure of the synthesized sultaine surfactant: 3-[didodecyl(2-h

3(

lm

act

2

2

seewficdbmrat1

(1

31

3

rpc

hydrophobic effect and can lead to a CVC value lower than that

ydroxyethyl)ammonio]-propane-1-sulfonate.

-[didodecyl(2-hydroxyethyl)ammonio]propane-1-sulfonateFig. 1), under controlled conditions of pH and osmolarity.

The structural properties of the polar headgroup and theipophilic region of this molecule suggest [1] the spontaneous for-

ation of vesicles in aqueous solution.The present work can contribute to the understanding of the

ggregation behaviour of sultaine surfactants in their solubilizationapabilities, stability and rheological properties for their prospec-ive employment as delivery systems.

. Experimental

.1. Synthesis

To obtain 2-didodecylaminoethanol in high yield (70%) a twotep alkylation of 2-aminoethanol with 1-bromododecane is nec-ssary. In the first step excess 2-aminoethanol was alkylated inthanol under reflux. The obtained ammonium salt was treatedith 0.1 M aqueous NaOH and the resulting precipitate was puri-ed by crystallization from hexane. The second alkylation wasarried out in ethanol and in the presence of Na2CO3. Crude 2-idodecylaminoethanol was purified by chromatography on SiO2y using a mixture of CH3OH/CHCl3 in the ratio 1/9 (v/v) as theobile phase. The final alkylation with 1,3-propanesultone under

eflux in 1,2-dicloroethane and the crystallization from an ethylcetate/acetone mixture led to the sultaine of Fig. 1. The identity ofhe surfactant (white powder, mp 135–137 ◦C,) was confirmed byH- and 13C-NMR spectroscopy (see Supplementary Materials):

ıH (300 MHz; CDCl3; Me4Si) 4.10 (2H, m), 3.75 (2H, m), 3.462H, m), 3.32–3.19 (4H, m), 2.96 (2H, m), 2.20 (2H, m), 1.66 (4H, m),.33–1.25 (36H, m), 0.90–0.85 (6H, t).

ıC (75 MHz, CDCl3; Me4Si) 60.96; 59.90; 58.78; 55.88; 47.87;2.24; 29.95; 29.81; 29.78; 29.68; 29.45; 26.73; 23.02; 22.24; 19.04;4.46.

. Materials

Pyrene, Nile Red, 5(6)-carboxyfluorescein (CF), sodium chlo-

ide, sucrose, acetone and ethanol (99% spectroscopic grade) wereurchased from Aldrich and Fluka and used without further purifi-ation.

B: Biointerfaces 87 (2011) 73– 78

3.1. Instruments

The fluorescence measurements were carried out by a Jasco FP-6200 spectrofluorimeter with a band pass of 5 nm.

The Dynamic Laser Light Scattering data were extrapolated byusing the Stokes–Einstein relationship for the calculation of thehydrodynamic radius from a Brookhaven (90PLUS BI-MAS) digi-tal correlator at a scattering angle of 90◦, equipped with a 35 mWHe–Ne laser at wavelength of 660 nm.

Dynamic and steady state rheological measurements were car-ried out on a Haake Mars II Thermoscientific rheometer with a coneand plate geometry (cone angle 0.5 rad, plate diameter 60 mm).

Video-enhanced microscopy measurements were carried out bya light inverse Zeiss Axiovert microscope, equipped with Hoffmannoptic 40× and a video camera for image detection.

DSC measurements were performed by using a Perkin ElmerDSC7 calorimeter. The thermogram was recorded between 30 and80 ◦C with a scan rate of 1.0 ◦C/min.

4. Methods

4.1. Sample preparation

1 mL of a 2 mM sultaine solution in chloroform was placed in aflask and the solvent was removed by evaporation under vacuumto obtain a dry film. Then 2 mL of a phosphate buffer solution of pH7.4 and 580 mOsm were added to the film under sonication.

The obtained suspension was stirred for 20 min and extrudedfive times at 60 ± 0.1 ◦C through polycarbonate filters (Osmonics,pore size 220 nm) mounted in a Lipex Biomembranes extruder(Vancouver BC, Canada). Unless otherwise stated, extrusion of thesultaine solutions was carried out before the experimental deter-minations due to its high reproducibility. After the extrusion theLUV solution was cooled to room temperature.

4.2. Determination of the CVC

Generally a surfactant with two long chains forms vesicles at aextremely low concentration referred as critical vesicular concen-tration (CVC) [19].

The determination of the CVC in phosphate buffer solution wasperformed as follows.

An aliquot of a 20 mM pyrene stock solution in ethanol wasadded to 2 mL of different sultaine buffered solutions under stirringat 25 ± 0.1 ◦C. The final concentration of pyrene in the samples was2 × 10−5 M. Each solution was left to equilibrate for 3 min beforerecording its fluorescence emission spectrum. It is well known[20,21] that the ratio of the first to the third vibronic peak I1/I3of a pyrene spectrum is a measure of the polarity of the mediumin which the probe is dissolved. Generally, in surfactant solutionsthe I1/I3 ratio decreases with increasing surfactant concentrationas an effect of aggregation, until it reaches a constant value lowerthan 1.0. A break in the curve obtained by plotting the I1/I3 valuesagainst the surfactant concentration clearly shows the formationof aggregates, depending upon the structure of the surfactant.The I1/I3 pyrene ratio also represents a useful tool for investigat-ing the bilayer mobility [22] and the integrity of the membrane[23]. The fluorescence emission spectra were taken at an excitationwavelength of 335 nm and I1 and I3 correspond to the emissionwavelength of 376 and 387 nm respectively. Since pyrene is hardlysoluble in water, it is shielded by surfactant molecules due to the

obtained in the absence of the probe [24,25].Nile Red has been widely employed [26] as a solvatochromic

fluorescent probe in monitoring surfactant aggregation, due to

P. De Maria et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 73– 78 75

Fs

igb[lgpotmcitpCC

tt

w

4

2wNpRbt

612

613

614

615

616

617

618

8070605040302010

T / °C

λm

ax / n

m

ig. 2. Micrograph of sultaine vesicles spontaneously formed in buffered aqueousolution before extrusion.

ts capability to highlight different morphologies for the aggre-ates formed by amphiphile molecules. This probe shows a largeathochromic absorbance shift with increasing solvent polarity27]. In aqueous solution, where Nile Red is poorly soluble, there is aarge preference of this probe to partition into the surfactant aggre-ates which offer hydrophobic binding sites. The locally decreasedolarity within the aggregates is expressed by an emission peakf the probe at wavelength lower than that in pure water. Hencehe critical aggregation concentration of a surfactant may be deter-

ined by following the emission peak as a function of the surfactantoncentration. To follow the shift in the maximum wavelengthnstead of the Nile Red fluorescence intensity has the advantagehat the CVC value is not influenced by the concentration of therobe and is free from the hydrophobic effect [28]. The sultaineVC values obtained by using pyrene have been compared to theVC values obtained by using Nile Red.

An aliquot of 2 mM Nile Red stock solution in acetone was addedo 2 mL of a series of buffered sultaine solutions. The final concen-ration of the probe in the samples was 3 × 10−6 M.

The wavelength used for the excitation in these experimentsas 490 nm.

.3. Temperature-dependent phase behaviour

10 �L of a 2 mM Nile Red solution in acetone was added to a mM sultaine solution and the emission fluorescence of the probeas determined in the temperature range 15–70 ◦C. The �max of theile Red fluorescence spectrum provides an indication about the

olarity of the environment in which the probe is located. Thus Nileed represents an useful probe for the determination of the phaseehaviour and for the transition among the different aggregateshat a surfactant can form.

0,9

1

1,1

1,2

1,3

1,4

-2,5-3-3,5-4-4,5-5-5,5-6

I 1/I 3

Log[Sultaine]

a b

Fig. 4. Fluorimetric determinations of the CVC values in

Fig. 3. Temperature-dependent phase behaviour of a 2 mM sultaine buffered solu-tion measured by Nile Red fluorescence.

4.4. Kinetic measurements

The dried film of the surfactant obtained after removal of CHCl3(as described above) was hydrated with 2 mL of a 50 mM buffered5(6)-carboxyfluorescein (CF) aqueous solution. The obtained sus-pension was extruded and run through a Sephadex G-50 column toremove the unentrapped dye from the loaded aggregates. The finalsultaine concentration was 3.6 × 10−5 M.

The stability of the vesicles was spectrofluorimetrically evalu-ated in the temperature range 25–75 ◦C at 580 mOsm by followingthe increase in fluorescence of CF at 516 nm (using 490 nm as theexcitation wavelength) due to the de-quenching of the entrappeddye upon release from vesicles.

The effect of an increase of the osmotic stress up to 1080 mOsmobtained by addition to the bulk solution of NaCl or sucrose wasevaluated kinetically at 70 ◦C by similarly following the release ofCF.

5. Results and discussion

The synthesized surfactant spontaneously forms vesicles with amean diameter of about 1 �m in buffered aqueous solution at pH

7.4 and 580 mOsm (Fig. 2).

The differential scanning calorimetry method used to determinethe temperature of phase transition Tm of sultaine gives a peaktransition at 45. 3 ◦C (see Supplementary Materials).

615

620

625

630

635

640

645

-2,5-3-3,5-4-4,5-5-5,5-6

λ max

/nm

Log[Sultaine]

buffer solution at 25 ◦C: (a) pyrene (b) Nile Red.

76 P. De Maria et al. / Colloids and Surfaces

300

350

400

450

500

550

600

650

0,0010,00080,00060,00040,00020

mea

n di

amet

er/n

m

[Sultaine]/M

F ◦

a

ig. 5. Mean diameter of the vesicles formed in a series of buffered solutions at 25 Ct different sultaine concentrations.

Fig. 6. Plot of G′ , G′′ and |�*| values for a 3.6 × 10−5 (a) and a 4.1 × 10−4

B: Biointerfaces 87 (2011) 73– 78

Above the Tm the alkyl chains of sultaine molecules convert fromgel to liquid–crystalline state.

The fluorescence emission �max of Nile Red inserted into sultainevesicles was measured in the temperature range 15–70 ◦C (Fig. 3).

The increase of Nile Red �max at high temperatures reflects theincrease in the polarity of the environment in which the probe islocated after its incorporation into the sultaine vesicles [26]. Thisbehaviour suggests the penetration of water between the head-groups of the sultaine molecules and is favoured by the formationof a less packed bilayers above 45 ◦C, according to the gel–liquidcrystalline transition obtained by DSC measurements.

These bilayer modifications associated with the well known pro-motion of contact regions between vesicles [29] upon increasingthe temperature could favour apposition of membranes [30] andexplain the vesicles aggregation evinced above 45 ◦C in the presentsultaine system.

Two different CVC values were observed in buffer solution afterthe extrusion above the Tm by using pyrene (Fig. 4a) and Nile Red(Fig. 4b) as the fluorescent probes. The two dyes are incorporatedinto vesicles as the sultaine molecules self-assemble.

Under these conditions the aggregation values are: CVC1

3.1 × 10−5 and 3.8 × 10−4 M in the case of pyrene; CVC2 2.8 × 10−5

and 6.4 × 10−4 M in the case of Nile Red. The values determined bymeans of the two different probes are in reasonable agreement.

M (b) sultaine solutions at 70 ◦C against the applied frequency.

P. De Maria et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 73– 78 77

Ft

s(l

aii

pw

cFmctst

tpat4u(uahdzaccv

gt

daogca

Table 1First order rate constants, kobs, for the spontaneous release of CF at differenttemperatures.

Temperature (◦C) 10−5 kobs (s−1)

25 ± 0.1 0.75 ± 0.0337 ± 0.1 1.30 ± 0.2550 ± 0.1 2.22 ± 0.0260 ± 0.1 2.53 ± 0.0465 ± 0.1 3.70 ± 0.17

An analogous leakage of CF cannot be observed above the CVC2.In fact, under these conditions, the sultaine solution becomes veryturbid and displays the above described visco-elastic behaviour,due to the aggregation of the vesicles where the dye resides.

Table 2First order rate constants, kobs, for the release of CF at 70 ◦C induced by osmoticstress.

Externalosmolarity at 70◦C

10−5 kobs (s−1) (±0.01)Sodium chloride

10−5 kobs (s−1) (±0.01)Sucrose

ig. 7. Spontaneous release of CF from sultaine vesicles at 25 ◦C. [Sul-aine] = 3.6 × 10−5 M.

From the different techniques used we obtain evidences thathow that the formation of vesicles occurs above the CVC13 × 10−5 M) and that, above the CVC2 (4 × 10−4 M), association intoarger polydisperse aggregates takes place.

Dynamic Laser Light Scattering measurements on the observedggregates have pointed out an increase in vesicle size rather thann the number of the aggregates as the sultaine concentrationncreases (Fig. 5).

This behaviour suggests that, at high concentration, closelyacked vesicles form, similar to the vesicles that DDAB forms inater in the presence of lecithin [16].

The presence of different type of aggregates at different con-entrations has also been investigated measuring by Oscillationrequency Sweep the dynamic viscosity, the elastic and viscousoduli of two different sultaine solutions at 25 and 70 ◦C. The con-

entration of the former solution, 3.6 × 10−5 M, was slightly abovehe CVC1 value, while that of the latter solution, 4.1 × 10−4 M, waslightly above the CVC2 measured with pyrene, but slightly belowhe value measured with Nile Red.

At 25 ◦C and a frequency of 10 Hz the measured values forhe 3.6 × 10−5 M solution are indistinguishable from those ofure water. This solution shows a liquid-like behaviour becauset this concentration sultaine LUV are small enough to avoidhe intervesicular interactions [31]. On the other hand, the.1 × 10−4 M solution displays low but accurately measurable val-es of the dynamic viscosity (|�*| = 0.01 Pa s) and an elastic modulusG′ = 12 Pa) 2 order of magnitude larger than the viscous mod-lus (G′′ = 0.07 Pa), typical of a gel-like system. Dispersions with

visco-elastic behaviour similar to that of the present systemave been previously observed for highly concentrated and dehy-rated onion-like vesicles [32], for multilamellar vesicles fromwitterionic surfactants/n-alcohols/ionic surfactants mixtures [33]s well as for decyltrimethylammonium hydroxides/2-hydroxy-1-arboxy-naphthoate systems [34]. This gel-like response at highoncentration indicates high interactions between close-packedesicles [35].

When the temperature is raised to 70 ◦C a typical visco-elasticel-like behaviour [36,37] has been observed for both sultaine solu-ions (Fig. 6).

At this temperature the dynamic viscosity is very high andecreases with increasing the applied frequency, while both modulire independent of frequency. A similar behaviour has been previ-

usly found in the case of multilamellar vesicles from tetraethylenelycol–monododecyl ether (C12E4) in D2O solution [38]. In thease of the 3.6 × 10−5 M sultaine solution this behaviour can bettributed to a change in the vesicles size due to their associa-

70 ± 0.1 5.60 ± 0.1975 ± 0.1 5.70 ± 0.13

tion and fusion induced by high temperature, while in the case ofthe 4.1 × 10−4 M solution this behaviour confirms the existence ofstable aggregated close-packed vesicles above the CVC2.

The spontaneous leakage of CF from the vesicles has been fol-lowed fluorimetrically at a concentration of about 3.6 × 10−5 M (i.e.slightly above the CVC1 of 3 × 10−5 M at 25 ◦C) in the temperaturerange 25–75 ◦C and at 580 mOsm. The process follows simple firstorder kinetics (a typical example is shown in Fig. 7), according tothe rate law of Eq. (1):

d[CF]dt

= kobs[CF] (1)

The obtained kobs values are reported in Table 1.The stability of the vesicles at 70 ◦C has also been studied under

hypertonic condition by adding sodium chloride or sucrose to thebulk solution. The obtained kobs values are reported in Table 2.

It is well known that the release of a dye from surfactant vesiclesis caused mainly by pores and defects in the bilayer [39,40]. Therate of release depends of temperature, osmotic imbalance and thepossible presence of guests incorporated into the bilayer and canbe increased by adding a surfactant as the perturbing agent [22].

The rate of CF efflux from the sultaine LUV increases withincreasing temperature as can be seen in Table 1. The process at70 ◦C is 7.5 times faster than at 25◦ and then, on further increasingthe temperature, the rate remains approximately constant.

The increase of CF leakage from the present sultaine LUV at hightemperature is probably due to the enhanced number of transientpores formed within the bilayers during membrane association.

The exposure of the LUV to the osmotic shock at 70 ◦C, imposedby increasing the osmolarity in the external solution from 580 to1080 mOsm, strongly decreases the rate of CF efflux (Table 2).

The presence of a membrane-impermeable small species in thebulk solution, such as NaCl or sucrose, produces a hydrostatic pres-sure gradient due to a mechanical imbalance across the bilayer andthe vesicles shrink in order to decrease their surface and compen-sate for the additional pressure.

During the shrinkage, some of the bilayer pores close [41] anda decrease in the aggregate size occurs. Consequently the stabilityof the LUV is enhanced, as shown by the observed decrease of therate of CF leakage.

(mOsm)

880 3.75 3.43980 4.22 3.34

1080 4.48 2.97

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8 P. De Maria et al. / Colloids and Su

ggregated vesicles do not break so that the hydrophilic probe canemain entrapped inside them for a considerable period of time.

. Conclusions

The new sultaine surfactant of Fig. 1 spontaneously forms vesi-les in buffered aqueous solution at pH 7.4 and 580 mOsm. Theransition temperature of about 45 ◦C has been found by DSC andonfirmed by fluorescence measurements. Two critical aggregationoncentrations have been measured, showing the existence of twoopulations of vesicles with different properties.

The stability of the extruded LUV formed above the CVC1ecreases from 25 to 75 ◦C as a consequence of membrane associa-ion and fusion among interacting vesicles. The addition of NaCl orucrose to the bulk solution induces a shrinkage of the vesicles thusmproving their stability at 70 ◦C. The same extruded LUV associatento larger closely packed aggregates at sultaine concentrationsbove the CVC2.

cknowledgments

This work has been supported by MIUR (PRIN 2008 prot.0085M2755) and Università “G. d’Annunzio” of Chieti-Pescara.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.colsurfb.2011.05.003.

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[[

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