Zwitterionic betaine transition from micelles to vesicles induced by cholesterol

    Zwitterionic betaine transition from micelles to vesicles induced by cholesterol R. Alenaizi, S. Radiman, I. Abdul Rahman, F. Mohamed PII: DOI: Reference:

S0167-7322(16)31022-4 doi: 10.1016/j.molliq.2016.09.062 MOLLIQ 6339

To appear in:

Journal of Molecular Liquids

Received date: Accepted date:

26 April 2016 20 September 2016

Please cite this article as: R. Alenaizi, S. Radiman, I. Abdul Rahman, F. Mohamed, Zwitterionic betaine transition from micelles to vesicles induced by cholesterol, Journal of Molecular Liquids (2016), doi: 10.1016/j.molliq.2016.09.062

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ACCEPTED MANUSCRIPT Zwitterionic betaine transition from micelles to vesicles induced by cholesterol R. Alenaizi a,*, S. Radiman a,b, I. Abdul Rahman a,b, F. Mohamed a,b

Nuclear Technology Research Center, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

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School of Applied Physics, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

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Vesicle formation by self-assembly in an aqueous mixture of zwitterionic carboxylate betaine surfactant and cholesterol was studied as a potential drug delivery nanocarrier. In this study, pyrene and 1,6-Diphenyle1-1,3,5-hexatrine (DPH) were applied as fluorescence probes that provides information on polarity and fluidity of the microenvironment, and used to monitor the transition of zwitterionic betaine from monomers to micelles, and to vesicles promoted by addition of cholesterol to micellar solutions through the variation of vibronic peak intensity ratio I1/I3 of the pyrene and solubilization with intensity quenching I428 of (DPH). In addition, the steady-state and time-resolved fluorescence anisotropy measurements of the (DPH) probe provides information on the effect of cholesterol on the dynamic properties vesicle membrane and vesicular distribution accompanied by dynamic light scattering (DLS), transmission electron microscopy (TEM) , and turbidity measurements. Our results show an abrupt change of the ratio I1/I3 of pyrene and fluorescence intensity I428 of (DPH) in the transition from monomer to micelle with increasing betaine concentration, as well as fluorescence anisotropy results thought the transition from micelle to vesicle promoted by cholesterol and confirmed by TEM result. Furthermore, the fluorescence anisotropy measurements shows that addition of small amount of cholesterol to zwitterionic betaine micelle have a significant influence on the molecular packing

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leading to vesicle formation and increasing bilayer rigidity with increasing cholesterol concentration up to χchol= 0.25.

Keywords: Vesicle; Zwitterionic surfactant; Betaine; Cholesterol; Anisotropy; Pyrene; DPH.

1. Introduction

Self-assembling amphiphiles have various shape and structures, such as micelles and vesicles in aqueous solution [1, 2]. Vesicles have wide range of applications, such as biomembrane and nano-carriers for hydrophilic and hydrophobic molecules in drug delivery systems [3-6]. Vesicles can be prepared by spontaneous self-assembly, mixed with co-surfactant or sterols such as cholesterol, and input of energy (e.g. sonication, homogenization, heating). In aqueous solution, the formation of unilamellar or multilamellar vesicles depends upon the concentration of lipid molecules, preparation methods, etc [7-9]. The

structural formation can be explained by the packing parameter theory [10, 11], since single-tailed surfactants tends to form micelles, while a doubletailed surfactants generally leads to the formation of bilayer structures in aqueous solution [1, 12, 13] . In the binary system of single-tailed surfactant, a morphological transition of aggregates is derived with suitable concentration and composition in the aqueous mixture, and is further promoted by addition of salt ions [13-15]. Liposomes are artificially created vesicles consisting of bilayers that may contain combinations of phospholipids and sterols [7, 16, 17]. The degradation of phospholipids by hydrolysis [18, 19], and the metastable structure of liposomes increases

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The transition from surfactant micelles to vesicles in aqueous solution is a unique phenomenon that mimics various biological processes, in generally the transition influenced by different external stimuli, such as polar additive, water insoluble sterol moieties, etc. [14, 18, 33-36]. Recently, many researchers reported the structure transition for single-tailed surfactant micelles promoted by addition of cholesterol to aqueous micellar solution. Veciana and coworkers [14, 33], they reported the detailed mechanisms regarding the formation of vesicles in aqueous solution of cationic surfactant, cetyltrimrthylammoniom bromide (CTAB) with increasing cholesterol content. While, Sarkar and coworkers [18, 19] reported cholesterol induced vesicle formation in aqueous solution of anionic surfactants, sodium dodecyl sulfate (SDS) and 1hexadecyl-3-methylimidazolium chloride

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([C16mim]cl), with increasing cholesterol content. Furthermore, cholesterol induced vesicle formation in aqueous of solution of nonionic surfactant, termed as niosomes, have been characterized using various nonionic surfactants molecules [37, 38]. However, to the best of our knowledge, the structural transition of inverted zwitterionic head group surfactant with single-tailed carboxylate betaine as shown in (Fig.1), in the presence of cholesterol has yet been reported. The carboxylate betaine is known to easily form micelles in the polar medium as water [39], and cholesterol alone cannot initiate bilayers formation, but interaction with zwitterionic betaine surfactant results in the assembly of vesicles exhibiting structural characteristic similar to those observed in liposomes [37]. Here we report data associated with vesicles formation obtained by self-assembly of single-tailed zwitterionic N-(Alkyl C10-C16)-N,N-dimethylglycine betaine and cholesterol. The structural transition from betaine monomer to micelle to vesicle mixture were monitored by fluorescence measurements with environment sensitive fluorescence membrane probes pyrene and 1,6-Diphenyle1-1,3,5-hexatrine (DPH). Furthermore, the steady-state and time-resolved fluorescence anisotropy measurements of (DPH) probe accompanied by dynamic light scattering (DLS), transmission electron microscopy (TEM), and turbidity measurements provides information on the effect of cholesterol on the dynamic properties vesicle membrane formation and vesicular distribution. Our results show an abrupt change of the fluorescence ratio I1/I3 of pyrene and fluorescence intensity quenching I428 and absorbance intensity ratio I1/I2 of (DPH) during the transition from monomers to micelles with increasing the concentration, as well as anisotropy results through the transition from micelles to vesicles promoted by cholesterol and it confirmed by TEM result. Furthermore, the anisotropy measurements shows the addition of small amount of cholesterol to zwitterionic betaine micelle have a significant influence on the molecular packing caused vesicle formation and increasing membrane rigidity with increasing cholesterol concentration.

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susceptibility to aggregation [20-22] restrict their usage as drug carriers and models for biological membrane. Therefore, there are increased interest to develop non-lipid vesicles by using surfactant molecules as alternative for phospholipids in order to enhance liposomal stability and functionality [18, 23]. The combined presence and orientation of both charges in the hydrophilic polar heads of zwitterionic phospholipids play an important role in the metastable structural transition, due to weak attractive forces in liposomes [24]. On other hand, lipids with inverted head group orientation have cationic moiety anchored at the membrane interface, with the anionic moiety extending into the aqueous phase. These lipids can form stable liposomes in the presence of cholesterol [25, 26]. Non-lipid vesicle bilayers formation can occur by a combination of surfactant aqueous mixtures along with cholesterol and salts; they have received much more attention compared to lipid vesicles due to better stability and easy preparation [15, 23, 27, 28]. Furthermore, cholesterol is among the important lipid species found in animal cell membranes, used for its ability to optimize certain physical properties of the cell membrane such as rigidity and fluidity of membranes [29-32].

ACCEPTED MANUSCRIPT (turbidity) is defined as the absorbance of an optical element at a given wavelength (λ) per unit of the element thickness [14, 40]. The wavelength selected to measure the optical density of zwitterionic betaine/cholesterol selfassembly system was λ=700 nm, at which none of the system elements absorb the light. All the turbidity measurements were performed at room temperature .

2. Experimental

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2.2. Sample preparation

2.4. Steady-State and Time-Resolved Fluorescence measurements

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In order to incorporate the fluorescence probes into the micelles stock solution, a small aliquot of each was weighed and dissolved in tetrahydrofuran (THF), separately. THF in the mixed solution was removed using nitrogen gas and later dried in vacuum at over night. The dried probes were suspended in 10ml of 20mM stock micellar solution of zwitterionic betaine surfactants each separately, and then mixed well by vortexing for 3min. The suspension was briefly sonicated by using (Wiseclean Ultrasonic Cleaner WUC-A03H, 40KHzand 124W) to obtain a homogenous stock micellar dispersion. The molar ratio of probe to surfactant was kept to 1:1000 mol% to give minimal perturbation to the micellar organization. Furthermore, to obtain betaine/cholesterol transition from micelle to vesicle by varying cholesterol concentrations, while keeping the betaine concentration at 20mM with DPH or pyrene in the same ratio. The appropriate amounts was weighed directly in glass tubes and suspended in 10ml of milli-Q water at a desired concentration. The mixtures were vortex mixed for 2 min, followed by 30 min. sonication using bath sonication at room temperature to produce vesicle dispersion. Before analysis, the dispersed samples were equilibrated at room temperature for 3 days. 2.3. UV-Vis and optical density

To obtain absorption spectra of DPH, a Perkin-Emler Lampda 35 UV-Vis spectrophotometer (Perkin-Elmer, USA) was used with 1-cm optical length matched pair quartz cuvette and performed at room temperature with a 2 nm slit width. The optical density

Fluorescence measurements with either pyrene or DPH as a probe were performed at with steady-state and time-resolved fluorescence spectrometer FLS920 (Edinburgh Instruments, UK), using 1 cm path length quartz cuvettes. The fluorescence intensity ratio I1/I3 of the first (I1, 373 nm) and third (I3, 384 nm) peaks in the spectra of pyrene were obtained with excitation at 335 nm. The samples containing DPH probe were excited at 357 nm and the emission was recorded in the wave length rang of 375600 nm and I428 was measured. Steady-state anisotropy measurements were taken with a polarization filter having L-format configuration to measure the fluorescence anisotropy rs of the DPH. A 375 nm cutoff filter was placed in the emission beam to eliminate the effects of scattered light, if any. The excitation and emission band width fixed at 0.5 nm. The anisotropy rs defined by

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N-(Alkyl C10-C16)-N,N-dimethylglycine betaine (EMPIGEN®BB detergent) with 35% active substance in H2O, 5-cholesten-3β-ol (Cholesterol with purity ≥ 99%), Pyrene with purity 98% and 1,6-Diphenyle1-1,3,5-hexatrine (DPH) with purity 98% used as fluorescence probes were purchased from Sigma-Aldrich, USA. These materials were used as received without any further purification. All experiments were conducted with pure deionized water that was passed through a Milli-Q Plus purification system with a resistivity of 18.2 MΩ·cm. The structure of the zwitterionic betaine, cholesterol, pyrene, and the DPH are shown in Fig.(1).

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2.1. Materials

(1) (2) Where G is the correction factor, IVV and IVH are the vertically and horizontally polarized emission intensities, respectively resulting from the vertically polarized excitation of the probe [13, 41]. The software supplied by the manufacture automatically determined the correction factor and anisotropy value. In all cases, the anisotropy values were averaged out for five measurements for each sample. A time correlated single photon counting (TCSPC) setup was used to collect time-resolved emission decay at the excitation wavelength of 376.4 nm with typical instrument response function at pulse width is 86 ps of (EPL-375, picoseconds pulsed diode laser). The time resolved fluorescence anisotropy is defined by the following equation:

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Vesicles were visualized using negative-staining TEM. A drop of vesicles dispersion was adsorbed onto a 200mesh copper grid coated with a formvar film and allowed to adhere following removal of excess liquid. After 5 minutes, a few drops of 2% Uranyl acetate solution was added to grid and left for 10 seconds prior to removal of excess solution The grid was then washed three times using distilled water, the sample air dried, and then visualized using a transmission electron microscope operated at 120 kV (Tecnai Biotwin, Netherland).

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For the anisotropy decays, we used a motorized polarizer in the emission side to collect the emission intensities at parallel IVV and perpendicular IVH polarizations at 430 nm emission maxima wavelength of DPH. The correction factor, G was estimated by using horizontally polarized excitation light. For our experimental setup, the value of the G factor was 0.55. The fluorescence anisotropy decay curves analyzed as a sum of exponential terms defined as:

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Where τir is a rotational time constant (lifetime) and ai is a pre-exponential factor representing the relative amplitude as . The decays were analyzed by using tail fit analysis of F900 decay analysis software. 2.5. Particle size distribution

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Zetasizer instrument (Nano-ZS, Malvern instruments, UK) was used and operated with a 4mW He-Ne ion laser, to obtain the hydrodynamic diameter (Dh) of self-assembly distribution of zwitterionic betaine/cholesterol in aqueous medium, and to monitor the effects of cholesterol on the aggregation distribution. The samples were measured directly without dilution and carried out at room temperature . The experimental results were performed in triplicate and presented as intensity of scattered light for aggregate distribution, as described in previous work [42].

Fig.1. The molecular structures of a) zwitterionic carboxylate betaine surfactant; b) cholesterol; C) 1,6-Diphenyle1-1,3,5hexatrine (DPH) and d) pyrene.

3. Results and discussion

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3.1 Zwitterionic formation

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It has been reported that fluorescence ratio I1/I3 of pyrene (the structure is shown in Fig.1) is related to the polarity of the pyrene environment [13, 40]. Low values of the ratio correspond to a nonpolar environment and it increases as the polarity of the environment rises. On other hand, DPH which was used as a hydrophobic fluorescence probe has a rodlike structure as shown in Fig.(1). It absorbs and emits light with a high quantum efficiency, composed of three vibronic peaks and shoulder corresponding to DPH in monomeric form [43-45] as shown in Fig.(2). The roughly mirrored spectra in Fig.(2) are attributed to the absorption corresponding to second excited state, while the fluorescence corresponds to first excited state [45-47]. Therefore, the ratio I1/I3 of pyrene and the solubilization with intensity quenching I428 and anisotropy values that were obtained by fluorescence polarization measurements of DPH, were employed as indicators of micropolarity and micro-viscosity, respectively [48, 49]. Hence, they can be used to monitor the transition of surfactants from monomers to micelles and vesicles assembly [50, 51]. In this work, fluorescence spectra of aqueous betaine surfactant solutions containing pyrene or DPH with betaine concentrations in the range 0.01 to 20 mM

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were recorded at . The change in the I1/I3 ratio of pyrene is shown in Fig.(3a). As pyrene binds to hydrophobic sites, the I1/I3 ratio is reduced by a factor characteristic of the particular microenvironment where the pyrene molecules are located. While at low concentration the betaine did not associates in aqueous solution, the I1/I3 value close to the ratio value of pyrene in water (I1/I3 ~2) as obtained separately, and DPH was not solubilized in hydrophobic environment, therefore, the fluorescence intensities I428 of DPH were very low as shown in Fig.(3b). At higher concentration betaine formed micelles and DPH were solubilized in the hydrophobic micelle interior, giving a characteristic spectrum. The inflection point of I1/I3 and I428 versus concentration curve corresponded to the CMC (~2 mM) of carboxylate betaine surfactant [39]. In this work, we report that DPH fluorescence and absorption intensities ratio I1/I2 are related to the polarity of the microenvironments around DPH, as well as in pyrene experience, with more preference to absorption characteristics as shown in Fig.(3c) since the second exited state of DPH has ionic characteristics and more sensitive to polarity of the ambient in the absorption process than the covalent fist exited state in fluorescence process [45]. Throughout the transition of surfactants from micellar to monomer under dilution process, the increase in vibronic peak intensities ratio I1/I3 of pyrene and I1/I2 of DPH indicated an increased polarity of ambient to probe, while the self-aggregation process of DPH in water (
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Fig.3. (a) fluorescence intensity ratio I1/I3 of pyrene, (b) fluorescence intensity I428 and (c) absorption intensity ratio I1/I2, of DPH as a function of betaine concentration in aqueous solutions.

Fig.2. Absorption (left) and fluorescence (right) spectra of DPH in zwitterionic betaine micellar solution.

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The phase transition of carboxylate betaine micelles to betaine/cholesterol vesicles by addition of cholesterol to involve successive formation of different intermediate supramolecular assemblies as suggested by the general mechanism of micelle to vesicle transition for surfactant/lipid systems [14, 53]. The self-assembly systems were prepared with varying cholesterol concentration while keeping betaine concentration constant above CMC at 20mM. Here the total concentration Ctot and the mole fraction Xchol of cholesterol are defined by

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3.2 Micelle to vesicle transition promoted by cholesterol

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The influence of the betaine /cholesterol molar ratio on the resulting organization is examined by steadystate and time-resolved fluorescence anisotropy measurements of DPH probe, dynamic light scattering DLS with turbidity measurements, and supported by transmission electron microscopy TEM results. In the mixture of betaine/cholesterol, pyrene and DPH also presents a fluorescence spectrum with high intensity since they are solubilized in the bilayer of the vesicle. The spectrum and intensities ratio I1/I3 of pyrene is much similar to that of micelles (I1/I3~1.3). To characterize the aggregate morphology in the mixture of betaine/cholesterol, the steady-state fluorescence anisotropy (rs) of DPH was used. The hydrophobic DPH probe has rod-like structure as shown in Fig.(1). Therefore, its rotational diffusion and their fluorescence anisotropy will be influenced by the fluidity of the hydrophobic microenvironment of the aggregates. The DPH has a long experience in such measurements, and register anisotropy value rs less than 0.14, while in bilayers or vesicles show rs value more than 0.14 [31, 41, 46, 50, 51].

Fig.4. Steady-state fluorescence anisotropy (a); Time-resolved fluorescence decays (b); variation of average rotation time (c), of DPH in betaine/cholesterol system as function to cholesterol molar fraction into 20mM aqueous betaine solution.

The time-resolved fluorescence anisotropy decays r(t) measurements were used to further understand the increased rigidity and hydrophobicity of the surrounding microenvironment of the probe through

ACCEPTED MANUSCRIPT Table 1. Fluorescence anisotropy decay parameters of DPH in aqueous solution of carboxylate betaine at different cholesterol mole fraction χchol.

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0 100 1.48 1.48 0.05 0.95 1.58 0.05 0.48 1.53 0.10 0.32 1.18 0.68 1.98 1.73 0.15 0.26 1.05 0.74 2.115 1.86 0.20 0.17 0.64 0.83 2.73 2.38 0.25 0.06 0.31 0.94 4.34 4.10 0.33 0.06 0.10 0.94 3.66 3.45 0.50 0.01 0.02 0.99 3.06 3.03 a <τr>= a1τ1r + a2τ2r for biexponential fluorescence decays where, τ1r and τ2r are rotational time constant with their corresponding relative amplitude а1 and а2.

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the micelle to vesicle transition at increasing cholesterol content. The anisotropy results are illustrated in Fig.(4), and shows in the absence of the cholesterol, betaine surfactant produced low steadystate anisotropy value (rs=0.065), and the anisotropy decay of DPH is single exponential, which was well with low local microviscosity (high fluidity) surrounding DPH molecules, which is comparable with viscosity profile and size distribution as shown in Fig.(7) for micelle structure. On other hand, addition of small amount of cholesterol to betaine micellar solution have a significant influence on the anisotropy value (rs≥0.25) as shown in Fig.(4a) where the anisotropy value increase sharply up to χchol=0.25 and then becomes saturated, which it was well with highly order microenvironment surrounding the DPH molecules, and it comparable with bilayers or vesicles anisotropy profile. Furthermore, Fig.(4b) show the anisotropy decay of DPH becomes gradually slower with increasing concentration of cholesterol in the micellar solution of carboxylate betaine with longer rotation relaxation time constant <τr> as shown in Fig.(4c), the anisotropy decays of DPH in all vesicular solutions were fitted well to biexponential function and the corresponding fitted decay parameters are given in table1 and depicted in Fig.(4c). The anisotropy decay and the rotation relaxation time reached the maximum up to χchol=0.25 which is in agreement with saturated steady-state anisotropy as shown in Fig.(4b) and Fig(4c), respectively. This observation confirms that as betaine micelles are transformed into vesicles upon addition of cholesterol and the bilayer becomes more order (rigid), the DPH molecules are incorporated into the rigid and confined microenvironments of the vesicles, and this is consistent with cholesterol effects in membrane order. The continuous increasing cholesterol concentration up to χchol=0.50 shows slightly decreases in anisotropy results, this indicates that the rotation of probe molecules depends upon the morphology, size, and structural heterogeneity of organized assemblies, and this is consistent with confirmed results of hydrodynamic diameter (dh) and turbidity variations as shown in Fig.(6) and Fig.(7).

Furthermore, stability of the assembly structures during dilution process was examined by using DPH probe. Fig.(5) shows the fluorescence spectrum for betaine surfactant at 20 mM concentration with/without cholesterol under dilution in excess water. It was clearly observed that the effect of the absence of the cholesterol on molecular assemblies, lead to considerable changes in the fluorescence spectrum at betaine concentrations lower than CMC (~2 mM). In contrast, the fluorescence spectrum of DPH remains unchanged in dilution until 1/100 of dilution for betaine/cholesterol system, indicating vesicular organization dominates in the presence of cholesterol, since the closed bilayers are more resistant to dissociate into monomers in dilution with water [14]. Also, the influence of cholesterol molar ratio on the resulting organization is monitored by turbidity and DLS measurements, and the results are illustrated in Fig.(6) and Fig.(7). In the absence of the cholesterol, the pure betaine micelle formed clear solution at concentration 20mM, while the dispersion exhibited bluish followed by more turbid appearance after gradually addition of cholesterol to micelles solutions. This variation in the turbidity of solutions is quite expected for micelle to vesicle transition, and the turbidity is found to be abruptly increased at (χchol> 0.25) due to the formation of larger aggregates. Furthermore, Fig.(7) shows the size distributions of carboxylate betaine/cholesterol aggregates at different

ACCEPTED MANUSCRIPT Since the light scattering intensity is proportional to the sixth power of the diameter of the particles we observed the major populations fastened around ~100 nm, while the minor population distributed around ~1000 nm due to formation large aggregation. These results are in agreement with the TEM results as depicted in Fig.(8), which are typical for vesicles structure, as reported in previous work [42]. The composition dependent of the turbidity and the size distribution are comparable with that of anisotropy in the support of the micelle to vesicle transition. Also the TEM measurements was directly supported vesicle formation induced by cholesterol as depicted in Fig.(8), the images indicated the formation of the spherical vesicles with average size in the range of 50200 nm.

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cholesterol molar fractions. In the absence of cholesterol, the major populations as obtained from DLS measurements is ~6 nm, which it is corresponding to micellar aggregation, another populations observed at (~500 nm) which is probably due to the formation of larger betaine aggregates. Similar results are also observed for the DLS measurements of the aqueous cationic surfactant (CTAB) [33], and anionic surfactant ([C16mim]Cl) [19]. The size distribution of the aggregates increases with increasing cholesterol content, and this is consistent with the micelle to vesicle transition.

Fig. 6. Optical density and average hydrodynamic diameter variation as function of cholesterol molar fraction into 20mM of betaine aqueous solution.

3. Conclusions

Fig.5. Normalized fluorescence spectrum of 20 µM DPH in (a) 20 mM micelle solution of carboxylate betaine surfactant, (b) 10:1 molar ratio of betaine/ cholesterol, at different betaine concentrations during the dilution.

The incorporation of cholesterol into the aqueous solutions of single-tailed zwitterionic carboxylate betaine surfactant results in formation of nanovesicle structures. Hence, molecular level investigations of composition dependence towards transition in diluted dispersion system are essential to overcome some of the limitations related to lipid vesicle systems. The transition from monomers to micelles, then from micelles to vesicles through varying mixing ratio of

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The anisotropy results shows that addition of small amount of cholesterol to betaine micelles have a significant influence on the molecular packing caused vesicles formation and increased bilayers rigidity with increasing cholesterol concentration. The measured of hydrodynamic diameters of the species with the variation in measured turbidity are comparable and supported the mixed vesicle formation during the transition, and confirmed the vesicle morphology by TEM micrograph images. Furthermore, their supramolecular organization of betaine/cholesterol appears to be rather stable with time and on dilution, offering alternative advantages for controlled drug delivery formulation.

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cholesterol was obtained from fluorescence measurements by a combination of two fluorescence probes, pyrene and DPH. The ratio of fluorescence intensity of pyrene I1/I3 and fluorescence intensity quenching I428 of DPH reflects the inner microenvironment polarity changes during the transition of betaine from monomers to micelles assembly. Also the absorption intensities ratio I1/I3 of DPH can be used to monitor the micro-polarity changes during the transition as well as pyrene experience. On other hand, the transition from micelles to vesicles induced by cholesterol obtained by steady-state and time-resolved fluorescence anisotropy measurements of DPH probe, accompanied by dynamic light scattering and turbidity measurements.

Fig. 7. Size distributions of carboxylate betaine/cholesterol forming supramolecular assemblies at different cholesterol molar fraction χchol.

Fig.8. Negative-staining TEM micrograph image of betaine/cholesterol system at different mole fraction of cholesterol a) χchol=0.10 and b) χchol=0.25, scale bar 200 nm.

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Acknowledgments The authors would like to thank universiti Kebangsaan Malaysia (UKM) for the financial support of this research work through the grant number UKM-DIP-2012-032, DLP-2013-037& DIP2014-022, ERGS/1/2012/STG02/UKM/02/1, AP2015-006.

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Šegota, S., Spontaneous formation of vesicles. Advances in colloid and interface science, 2006. 121(1): p. 51-75. Chen, W.-J., et al., Spontaneous vesicle formation and vesicle–tubular microstructure transition in aqueous solution of a poly-tailed cationic and anionic surfactants mixture. Journal of colloid and interface science, 2004. 278(2): p. 447-452. Jin, Q., et al., Zwitterionic drug nanocarriers: A biomimetic strategy for drug delivery. Colloids and Surfaces B: Biointerfaces, 2014. 124: p. 80-86. Al-Jamal, W.T. and K. Kostarelos, Liposomes: from a clinically established drug delivery system to a nanoparticle platform for theranostic nanomedicine. Accounts of chemical research, 2011. 44(10): p. 10941104. Saraf, S., Applications of novel drug delivery system for herbal formulations. Fitoterapia, 2010. 81(7): p. 680-689. Brandl, M., Liposomes as drug carriers: a technological approach. Biotechnology annual review, 2001. 7: p. 59-85. Laouini, A., et al., Preparation, characterization and applications of liposomes: state of the art. Journal of Colloid Science and Biotechnology, 2012. 1(2): p. 147-168. Akbarzadeh, A., et al., Liposome: classification, preparation, and applications. Nanoscale Res Lett, 2013. 8(1): p. 102.

12.

NU

References 1.

RI

PT

10.

Mozafari, M.R., Liposomes: an overview of manufacturing techniques. Cellular and Molecular Biology Letters, 2005. 10(4): p. 711. Israelachvili, J.N., D.J. Mitchell, and B.W. Ninham, Theory of self-assembly of hydrocarbon amphiphiles into micelles and bilayers. Journal of the Chemical Society, Faraday Transactions 2: Molecular and Chemical Physics, 1976. 72: p. 1525-1568. Tanford, C., Micelle shape and size. The Journal of Physical Chemistry, 1972. 76(21): p. 3020-3024. Antonietti, M. and S. Förster, Vesicles and liposomes: a self‐assembly principle beyond lipids. Advanced Materials, 2003. 15(16): p. 1323-1333. Kodama, T., et al., Fluorescence-probe study of vesicle and micelle formations in a binary cationic surfactants system. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2006. 277(1): p. 20-26. Cano-Sarabia, M., et al., Cholesterol induced CTAB micelle-to-vesicle phase transitions. Journal of colloid and interface science, 2010. 350(1): p. 10-15. Zhai, L., et al., Influence of salt and polymer on the critical vesicle concentration in aqueous mixture of zwitterionic/anionic surfactants. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2006. 276(1): p. 28-33. Mannock, D.A., et al., The effect of variations in phospholipid and sterol structure on the nature of lipid–sterol interactions in lipid bilayer model membranes. Chemistry and Physics of Lipids, 2010. 163(6): p. 403-448. Huang, J., J.T. Buboltz, and G.W. Feigenson, Maximum solubility of cholesterol in phosphatidylcholine and phosphatidylethanolamine bilayers. Biochimica et Biophysica Acta (BBA) Biomembranes, 1999. 1417(1): p. 89-100. Ghosh, S., et al., How does the surface charge of ionic surfactant and cholesterol forming vesicles control rotational and translational motion of rhodamine 6G perchlorate (R6G ClO4)? Langmuir, 2015. 31(8): p. 2310-2320.

13.

14.

15.

16.

17.

18.

ACCEPTED MANUSCRIPT

22.

25.

26.

27.

28.

TE

AC CE P

24.

PT

RI

31.

32.

33.

D

23.

30.

Journal of Colloid and Interface Science, 2006. 295(1): p. 243-248. Róg, T., et al., Ordering effects of cholesterol and its analogues. Biochimica et Biophysica Acta (BBA) - Biomembranes, 2009. 1788(1): p. 97-121. de Meyer, F. and B. Smit, Effect of cholesterol on the structure of a phospholipid bilayer. Proceedings of the National Academy of Sciences, 2009. 106(10): p. 3654-3658. Shrivastava, S. and A. Chattopadhyay, Influence of cholesterol and ergosterol on membrane dynamics using different fluorescent reporter probes. Biochemical and biophysical research communications, 2007. 356(3): p. 705-710. Shrivastava, S., et al., Differential Effects of Cholesterol and its Immediate Biosynthetic Precursors on Membrane Organization†. Biochemistry, 2008. 47(20): p. 5668-5677. Ferrer-Tasies, L., et al., Quatsomes: Vesicles formed by self-assembly of sterols and quaternary ammonium surfactants. Langmuir, 2013. 29(22): p. 6519-6528. Li, W., et al., CO 2-induced micelle to vesicle transition in zwitterionic–anionic surfactant systems. Physical Chemistry Chemical Physics, 2014. 16(8): p. 3640-3647. Jiang, L., et al., Bile salt-induced vesicle-tomicelle transition in catanionic surfactant systems: steric and electrostatic interactions. Langmuir, 2008. 24(9): p. 4600-4606. Yin, H., et al., Micelle‐to‐Vesicle Transition Induced by Organic Additives in Catanionic Surfactant Systems. Chemistry–A European Journal, 2006. 12(10): p. 2825-2835. Manosroi, A., et al., Characterization of vesicles prepared with various non-ionic surfactants mixed with cholesterol. Colloids and Surfaces B: Biointerfaces, 2003. 30(1): p. 129-138. Mandal, S., et al., Modulation of the photophysical properties of curcumin in nonionic surfactant (Tween-20) forming micelles and niosomes: a comparative study of different microenvironments. The Journal of Physical Chemistry B, 2013. 117(23): p. 6957-6968.

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29.

NU

20.

Mandal, S., et al., Vesicles Formed in Aqueous Mixtures of Cholesterol and Imidazolium Surface Active Ionic Liquid: A Comparison with Common Cationic Surfactant by Water Dynamics. The Journal of Physical Chemistry B, 2014. 118(22): p. 5913-5923. Sharma, A. and U.S. Sharma, Liposomes in drug delivery: progress and limitations. International journal of pharmaceutics, 1997. 154(2): p. 123-140. Marques, E.F., Size and stability of catanionic vesicles: effects of formation path, sonication, and aging. Langmuir, 2000. 16(11): p. 47984807. Liu, D.-Z., et al., Microcalorimetric and shear studies on the effects of cholesterol on the physical stability of lipid vesicles. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2000. 172(1): p. 57-67. Mondal, S., et al., The cholesterol aided micelle to vesicle transition of a cationic gemini surfactant (14-4-14) in aqueous medium. RSC ADVANCES, 2016. 6(31): p. 26019-26025. Park, Y., et al., Colloidal dispersion stability of unilamellar DPPC vesicles in aqueous electrolyte solutions and comparisons to predictions of the DLVO theory. Journal of Colloid and Interface Science, 2010. 342(2): p. 300-310. Kohli, A.G., C.L. Walsh, and F.C. Szoka, Synthesis and characterization of betaine-like diacyl lipids: zwitterionic lipids with the cationic amine at the bilayer interface. Chemistry and Physics of Lipids, 2012. 165(2): p. 252-259. Perttu, E.K. and F.C. Szoka, Zwitterionic sulfobetaine lipids that form vesicles with salt-dependent thermotropic properties. Chemical Communications, 2011. 47(47): p. 12613-12615. Carafa, M., et al., Preparation and properties of new unilamellar non-ionic/ionic surfactant vesicles. International Journal of Pharmaceutics, 1998. 160(1): p. 51-59. McLachlan, A.A. and D.G. Marangoni, Interactions between zwitterionic and conventional anionic and cationic surfactants.

MA

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35.

36.

37.

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ACCEPTED MANUSCRIPT

44.

45.

46.

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(oxyethylene-b-oxypropylene-b-oxyethylene) solutions. Macromolecules, 1995. 28(7): p. 2303-2314. Lin, Y. and P. Alexandridis, Self-assembly of an amphiphilic siloxane graft copolymer in water. The Journal of Physical Chemistry B, 2002. 106(42): p. 10845-10853. Subuddhi, U. and A.K. Mishra, Micellization of bile salts in aqueous medium: a fluorescence study. Colloids and Surfaces B: Biointerfaces, 2007. 57(1): p. 102-107. Zhang, X., J.K. Jackson, and H.M. Burt, Determination of surfactant critical micelle concentration by a novel fluorescence depolarization technique. Journal of biochemical and biophysical methods, 1996. 31(3): p. 145-150. Gracetto, A.C., et al., Self-aggregation processes of 1, 6-diphenyl-1, 3, 5-hexatriene in water/ethanol mixtures with high water percentages. Applied spectroscopy, 2011. 65(6): p. 604-610. Ollivon, M., et al., Vesicle reconstitution from lipid–detergent mixed micelles. Biochimica et Biophysica Acta (BBA)-Biomembranes, 2000. 1508(1): p. 34-50.

SC

41.

49.

51.

NU

40.

Weers, J.G., et al., Effect of the intramolecular charge separation distance on the solution properties of betaines and sulfobetaines. Langmuir, 1991. 7(5): p. 854-867. Zhai, L., et al., Transition from micelle to vesicle in aqueous mixtures of anionic/zwitterionic surfactants studied by fluorescence, conductivity, and turbidity methods. Journal of colloid and interface science, 2005. 284(2): p. 698-703. Pan, A., et al., Synergism between anionic double tail and zwitterionic single tail surfactants in the formation of mixed micelles and vesicles, and use of the micelle templates for the synthesis of nano-structured gold particles. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2015. 481: p. 644-654. Alenaizi, R., et al., Zwitterionic betainecholesterol system: Effects of sonication duration and aging on vesicles stability. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2015. 482: p. 662-669. Illinger, D., et al., A comparison of the fluorescence properties of TMA-DPH as a probe for plasma membrane and for endocytic membrane. Biochimica et Biophysica Acta (BBA)-Biomembranes, 1995. 1239(1): p. 58-66. Dupuy, B. and M. Montagu, Spectral properties of a fluorescent probe, all-trans-1, 6-diphenyl-1, 3, 5-hexatriene. Solvent and temperature effects. Analyst, 1997. 122(8): p. 783-786. Gracetto, A.C., et al., Unusual 1, 6-diphenyl-1, 3, 5-hexatriene (DPH) spectrophotometric behavior in water/ethanol and water/DMSO mixtures. Journal of the Brazilian Chemical Society, 2010. 21(8): p. 1497-1502. Lentz, B.R., Membrane “fluidity” as detected by diphenylhexatriene probes. Chemistry and Physics of Lipids, 1989. 50(3): p. 171-190. Saltiel, J. and S. Wang, Absorption and fluorescence spectra of a rigid analogue of all-trans-1, 6-diphenyl-1, 3, 5-hexatriene. J. Am. Chem. Soc, 1995. 117(10): p. 761-10. Kabanov, A.V., et al., Micelle formation and solubilization of fluorescent probes in poly

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Zwitterionic betaine transition from micelles to vesicles promoted by cholesterol. Cholesterol has a significant influence on the molecular packing of betaine vesicles. Measured fluorescence anisotropy describes cholesterol effects in bilayers rigidity. Supramolecular organization of betaine/cholesterol appears to be stable with time.

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