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Colloids and Surfaces A

journal homepage: www.elsevier.com/locate/colsurfa

Microstructural transitions in β-carotene loaded nonionic microemulsionsupon aqueous phase dilutionJing Guoa,b, Long Zhanga, Yuanyou Wangc, Tianqing Liua,⁎, Xiaolong Guba School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou, 225002, Chinab School of Biological and Chemical Engineering, Yangzhou Polytechnic College, Yangzhou, 225009, Chinac Department of Chemical Engineering, Yangzhou Polytechnic Institute, Yangzhou, 225127, China

G R A P H I C A L A B S T R A C T

A R T I C L E I N F O

Keywords:Biocompatible microemulsionβ-caroteneMicrostructural transitionsAqueousPhase dilution

A B S T R A C T

In this study, biocompatible formulation of a fully dilutable microemulsion system comprising Tween 60/iso-propyl myristate/ethanol/glycerol/water has been accomplished for solubilizing a natural antioxidant, β-car-otene. Solubilization capacity of β-carotene in the microemulsion system decreases progressively with increaseof aqueous phase weight fraction (Φ). Structural transitions occurring in the free and solubilized microemulsionswere investigated and compared via multiple complementary characterization methods. The characterizationhas been carried out by means of conductivity, viscosity, small angle X-ray scattering (SAXS), surface tension andtransmission electron microscopy. The results show that along the selected dilution line, the microemulsionundergoes a continuous transition from water-in-oil (W/O) micelles to oil-in-water (O/W) microemulsion via anordered bicontinuous structure. The β-carotene loaded has little influence on either the size or the shape ofmicro-domains or microstructural transitions in the microemulsions. Furthermore, the location of the β-carotenemolecules in the microemulsion system varies with the microstructural transition upon dilution of the aqueousphase. The detailed understanding of the structural behavior of the microemuslion systems might be significantfor their future applications in drug or nutraceutical delivery.

https://doi.org/10.1016/j.colsurfa.2019.01.041Received 30 July 2018; Received in revised form 22 January 2019; Accepted 22 January 2019

⁎ Corresponding author.E-mail address: tqliu@yzu.edu.cn (T. Liu).

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Available online 23 January 20190927-7757/ © 2019 Elsevier B.V. All rights reserved.

T

1. Introduction

Surfactants are well known to self-assemble into a wide range ofaggregates, including micelles, microemulsions, emulsions, vesicles,etc., which have received a growing quantity of attention in researchand industry fields [1–4]. Among the numerous colloidal assemblies,microemulsions have aroused intensified interest in pharmaceutical andcosmetic fields due to their ease of preparation, unique physicochem-ical properties, biocompatibility and long-lived stabilization. Micro-emulsions are optically isotropic and thermodynamically stable dis-persions of oil and water stabilized by an interfacial film of suitablesurfactant(s) usually in combination with a co-surfactant [5]. Becauseof the ultra-low interfacial tension, nanometric size (10˜200 nm) andhigh solubilization capacity, microemulsions frequently serve as de-livery vehicles for transporting drug and/or bioactive moleculesthrough human membranes [5,6]. Moreover, a prominent feature ofmicroemulsions in structural perspective is the variability of theirmicro-domains, such as water-in-oil (W/O), oil-in-water (O/W) andbicontinuous (B.C.) structures, which are formed depending on thecurvature and elasticity of the interfacial film [7,8]. It is essential toidentify the structural types of microemulsions and structural transi-tions occurring in the systems with dilution and/or in presence of anadditive, which directly affect practical applications of microemulsionsystems concerning additives solubilization and release.

β-carotene, one of the most commonly available carotenoids innature, exhibits preventive and therapeutic effects in major disorderslike cancer, eye problem, cardiovascular disease, light-induced er-ythema and other age related diseases [9,10]. Because of its capacity toquench singlet oxygen (1O2) and scavenge free radicals [11], β-caroteneis much attractive in developing health care medicine and skin careproducts. However, the physicochemical properties of β-carotene,particularly its poor water solubility and chemical instability, limit itsutilization as a nutraceutical ingredient in pharmaceutical and cosmeticformulations. Therefore, considerable work in selecting proper deliveryvehicles of β-carotene has been devoted, including O/W nanoemulsions[12,13], nanodispersions [14,15], nanostructured lipid particles [16]and liposomes [17]. These aforementioned delivery systems for β-car-otene were mainly involved in food processing aspects, and they werenot thermodynamically stable, thus inevitably undergoing destabiliza-tion. Garti and co-workers [18,19] successfully constructed food grademicroemulsions capable of solubilizing carotenoids and investigated theeffects of different factors on the solubilization capacity. Lopes et al.[20] described the utility of microemulsions as topical delivery vehiclesand examined skin penetration and antioxidant activity of lycopene inmicroemulsion vehicle. However, the effect of carotenoids on micro-emulsion structure and structural transitions, which has much to dowith the bioavailability of the additives, was rarely involved.

It is known that toxicity of all the constituting components of mi-croemulsion is one of the most important factors affecting its applica-tion in actives delivery aspects. The constituents are therefore needed tobe biologically and pharmaceutically acceptable, particularly the sur-factant and cosurfactant. To this end, we attempted to construct abiocompatible microemulsion system, for bioactive matter β-carotene,comprising Tween 60 (innocuous, nonionic biological surfactant [21]),isopropyl myristate (commonly used in cosmetics and pharmaceutics[22,23]) and short chain alcohols (i.e. ethanol and glycerol). To explorethe delivery potential of the constructed systems for actives, we focusedon the formation of U-type, fully dilutable with aqueous phase, Tweenbased microemulsions and characterization of the microstructuraltransitions in empty and β-carotene-loaded microemulsions. The mi-crostructure changes occurring in the systems were investigated by thecombination of multiple complementary techniques, including con-ductivity, viscosity, surface tension, small angle X-ray scattering (SAXS)and electron microscopic analyses. The objectives of our study are tofind out how the β-carotene molecules affect the structure and prop-erties of the microemulsion system, thereby speculating the location of

β-carotene in different types of the microemulsion. It is expected thatthe findings will provide a convenience for comprehensively detectingmicrostructural transitions in microemulsions and improve our under-standing on the microstructure of such systems acting as drug and ac-tives carriers. For application perspective, our findings will potentiallyserve as a basis for the research into encapsulation and release of li-pophilic nutraceuticals or pharmaceuticals.

2. Materials and methods

2.1. Materials

β-carotene (≥96% purity, Fig. S1) and isopropyl myristate (IPM)were purchased from Aladdin Biochemical Technology Co., Ltd.(Shanghai, P. R. China). Tween 60 [polyoxyethylene (20) sorbitanmonostearate], ethanol (EtOH) and glycerol were obtained fromSinopharm Chemical Reagent Co., Ltd (Shanghai, P. R. China). Thewater used was purified by an ultra-pure water purifier (Barnstead LabTower EDI 15, Thermo Scientific, USA).

2.2. Microemulsion preparation and phase behavior

Microemulsions were prepared by adding the aqueous phase com-prising of water and glycerol to an empty concentrate, i.e. a ternarymixture of Tween 60, IPM and ethanol. The samples were kept at25 ± 0.1 ℃ and monitored visually every day for a month.Transparent and monophasic samples were designated as microemul-sions, while opalescent or turbid ones followed by phase separationwere considered as multiphasic solutions. All the phases observed weremapped in pseudo-ternary phase diagram. The β-carotene-loaded con-centrate was prepared by solubilization of 150 ppm β-carotene in theempty concentrate. Each microemulsion sample was prepared in ascrew-capped glass vial and mixed by a vortex mixer (ScientificIndustries, USA) until a transparent solution was obtained. All mea-surements were carried out after resting the desired samples for 24 h inorder to ensure an equilibrium state was reached.

2.3. Solubilization measurements

A ready-made microemulsion was added to a glass vial into whichappropriate amounts of β-carotene had been deposited previously. Thiswas done by placing a stock β-carotene solution in dichloromethane inthe vial and by further evaporating the organic solvent under a streamof nitrogen. The appearance of turbidity or precipitates means that themicroemulsion sample was saturated or supersaturated. The cumulativeamount of added β-carotene before the saturated phenomena is used forthe quantification of the maximum solubilization capacity of β-car-otene.

2.4. Conductivity measurements

The electrical conductivity was measured by a digital conductivitymeter (model DDS-11 A, Shanghai Rex Xinjing Instrument Co., Ltd.,China). Measurements were performed on the microemulsion samplesupon dilution with aqueous phase up to 95wt% (along the dilution lineL64, in Fig. 2). The electrode was dipped into the microemulsionsample until equilibrium was reached and reading was stable. Thetemperature was kept at 25 ± 0.1 ℃ and maintained by a water baththermostat. The constant of conductivity cell was calibrated using0.01M KCl solution.

2.5. Viscosity measurements

Viscosity measurements were performed on the microemulsionsamples by a Rheostress 600 rheometer (Thermo Haake Corporation,Germany) with a DG41 double-slit rotor (the slit distance was 5.1mm).

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Shear rates were from 0.01 to 1500 s−1 and the temperature wascontrolled to 25 ± 0.1 ℃. The measurements were repeated threetimes and the results were found to be reproducible.

2.6. SAXS measurements

Scattering experiments were performed using a NanoStar SAXSequipped with a two-dimensional position sensitive detector (VÅNTEC-2000, Bruker AXS, Germany). The X-ray beam of Cu Kα radiation(λ=1.54 Ả) was further monochromated by a cross-coupled Göbelmirror. The sample was held in a 2mm quartz capillary and then flame-sealed. The measurements were performed at a constant temperature of25 ± 0.1 ℃. Sample-to-detector distance of 1070mm was selected tocover a range of scattering vector q from 0.07 to 2.30 nm−1.

2.7. Analysis of SAXS data

The scattering patterns were recorded and integrated into scatteringintensities I (q) depending on the scattering vector q = (4π/λ) sin θ,where λ is the wavelength of X-rays and 2θ is the scattering angle. Afterbackground subtraction, the scattering patterns for all values of q werefit to the expression proposed by Teubner and Strey [24]:

=+ +

+I qa c q c q

b( ) 12 1

22

4 (1)

where the constants a2, c1, c2, b were obtained by nonlinear leastsquares fitting of the experimental data. Eq. (1) corresponds to a realspace correlation function as follows:

=r krkr

e( ) sin r/(2)

This correlation function describes a structure with periodicity d(= 2π/k) damped as a function of correlation length ξ [25]. Values of dand ξ are related to the constants in Eq. (1) and given by [24]:

=k ac

cc

[ 12 4

]2

2

1/21

2

1/2(3)

= +ac

cc

[ 12 4

]2

2

1/21

2

1/2(4)

2.8. Surface tension measurements

Surface tension was measured at 25 ± 0.1 ℃ with an optical con-tact angle measuring device (OCA40, Dataphysics Instrument Co., Ltd.,Germany) using pendant drop method. The measurements were per-formed after calibration of the instrument with deionized water(72.00 ± 0.20mN·m−1) and repeated in triple to obtain mean values.

2.9. Transmission Electron Microscopy (TEM) measurements

The microstructure and morphology of the microemulsion sampleswere observed using Tecnai 12 transmission electron microscope(Philips Co., Netherlands). A drop of the sample was added onto acopper grid and equilibrated for 10min. The grid was then blotted withfilter paper to remove excess liquid and stained with 2% phospho-tungstic acid solution. Finally, the grid was transferred to the micro-scope (equipped with a Gatan 792 CCD camera), operating at an ac-celeration voltage of 120 kV.

3. Results and discussion

3.1. Phase behavior of Tween60/IPM/EtOH/glycerol/water system

Fig. 1 shows the phase diagram of the system composed ofTween60/IPM/EtOH/glycerol/water at 25 ± 0.1 ℃. The phase

behavior, as shown in Fig. 1, exhibits a large single phase and isotropicmicroemulsion region combined with multiphase regions appearing inboth sides of the phase diagram. L64 line indicates that a mixture ofIPM and EtOH at a constant weight ratio of 1:3 is mixed with Tween60,at a fixed weight ratio of 4:6, to form a transparent concentrate which isdiluted by aqueous phase comprising of water and glycerol at 1:1 wtratio. We selected infinite dilution line L64 for all measurements be-cause the system based on L55 is unstable, particularly at high aqueousphase concentration and the system based on L73 rich in surfactant isnot recommended (Fig. 1). Therefore, the concentrates based on L64,empty and loaded with 150 ppm β-carotene, were prepared and diluted.The physical characteristics of these systems were examined and elu-cidated.

3.2. Solubilization capacity of β-carotene

The microemulsion system prepared along the dilution line L64(Fig. 1) was selected to evaluate β-carotene solubilization capacity. Theamount (μg/g microemulsion) of β-carotene solubilized in the micro-emulsion is defined as solubilization capacity (SC). The variation of SCas a function of aqueous phase content (Φ) is presented in Fig. 2. FromFig. 2, it is clearly evident that the maximum SC of β-carotene decreaseswith increasing Φ and the decreasing degree is inconsistent at differentdilution range. As Φ increases to 20wt%, the SC steeply decreases from653 to 371 μg/g microemulsion. Upon further dilution, the SC

Fig. 1. Pseudo-ternary phase diagram of Tween 60/IPM/EtOH/glycerol/watersystem at 25 ± 0.1℃. The weight ratios of IPM/EtOH and water/glycerol werekept constant at 1/3 and 1/1, respectively. The line L64 represents the aqueousphase dilution line along which all the experiments were carried out. Single-phase (microemulsion) and multiphase regions are represented by I and II.

Fig. 2. Solubilization capacity (SC) curves of β-carotene along the dilution lineL64 in the system presented in Fig. 1. Inset: The calculated IF values as afunction of aqueous phase weight content (Φ).

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continues to decrease, but slowly as enough. As Φ exceeds 60wt%, theSC sharply decreases once more and tends to decrease moderately upondilution to 80wt%.

The above changes of SC upon aqueous phase dilution may resultfrom several factors, such as the dilution with aqueous phase, inter-facial packing of β-carotene and so on. To see whether the change of SCis dominated by the surfactant interface or the aqueous phase dilution,three different parameters (CFΦn, DFΦn and IFΦn) are calculated ac-cording to the SC values [23,26]. The three parameters are termed inturn as solubilization capacity change factor, dilution factor and overallinterfacial contribution factor. They can be calculated from the fol-lowing equations [23]:

=CF SCSC

nn

n 1 (5)

=DF 11

n n

n 1 (6)

=IF CFDF

nn

n (7)

where Φn denotes each aqueous phase weight content, and accordingly,Φn-1 denotes the previous aqueous phase weight content. The IF valuecan indirectly signify the role that surfactant interface played in thevariation of solubilization capacity. When IFΦn﹤1, the dilution factorDFΦn is dominant and the interface does little for the solubilization,which suggest that the solubilizate resides mainly in the oil phase. AsIFΦn=1, the interface and dilution factors contribute equally, in-dicating that the solubilizate is located partly at the interface and partlyin the oil phase. Correspondingly, once IFΦn﹥1, the interface plays adecisive role and thus the solubilizate is mostly embedded at the in-terface [23,26]. In brief, the more IF value increases, the more im-portant role the interface plays in the solubilization capacity.

From the inset of Fig. 2, the solubilization process of β-carotenemolecules can be reflected by variations of the IF values. Initially, thecalculated IF value is below 1, but increases gradually with the increaseof Φ. As Φ exceeds 20wt%, the IF value turns to be above 1. Thephenomena suggest that the β-carotene molecules are mostly located inthe oil phase at lower aqueous phase content (Φ﹤20 wt%) and thengradually move into the interface owing to the addition of the aqueousphase. Upon dilution to 50wt% aqueous phase, the IF value reaches toits maximum. However, the value decreases rapidly with further dilu-tion and regains to be less than 1 as Φ exceeds 60wt%, which indicatethat more and more β-carotene molecules transfer from the interface tothe oil phase in the dilution range of 50˜70wt% aqueous phase. Afterpassing through the minimum, the IF value increases once again withincreasing Φ and surpasses 1 at 90 wt% aqueous phase. It can be con-cluded that most β-carotene molecules will solubilize in the surfactantinterface when the water content is high enough. The dynamic varia-tion of β-carotene solubilization could indicate gradual microstructuretransitions occurring in the microemulsions. In order to further studythe microstructural changes in the free and loaded microemulsions andto determine the effect of the solubilized β-carotene on the micro-structure, we adopted multiple techniques, including conductivity,viscosity, surface tension, SAXS and TEM.

3.3. Conductivity and microstructural transition

Conductivity measurement turns out to be an effective approachwhich can provide an useful information of microstructural transitionsoccurring in microemulsion systems [27,28]. The continuous phase ofmicroemulsion can be estimated by the conductivity values (σ). Fornonionic microemulsion at lower water content, σ is similar to that ofthe oil phase, while at higher water content, it has similar σ to that ofpure water [29–32]. Fig. 3a shows variations of σ as a function ofaqueous phase weight content, Φ, for empty and β-carotene-loaded

microemulsions. At very low aqueous phase content, σ is negligible,which suggests that the isolated droplets in non-conducting oilymedium have little interactions with each other. When Φ exceeds 15wt%, σ starts to increase, but only marginally. In this region, a smallamount of water domains collide successively, which leads to a tran-sient formation of conductive channels and explains the moderate in-crease of the conductivity. After the aqueous phase dilution reaches Φp

(˜20 wt% aqueous phase), a steep and linear increase of conductivity isobserved (Fig. 3a), which is attributed to the occurrence of percolationbehavior [32]. This sharp increase of conductivity indicates that theaddition of aqueous phase makes W/O micelles swollen. A sticky col-lision between the water droplets leads to the droplets fusion, andtherein the conductive channels are well developed. However, when Φincreases up to ˜60 wt%, σ rises gently to the maximum value (Φ=75wt%), which can be explained that the discrete droplets turn intoa network of conductive channel and the B.C. structure is formed[32,33]. From then on, σ decreases gradually with the addition ofaqueous phase, which means large amount of water becomes the con-tinuous phase and the head groups of the surfactant are fully hydrated,with a structural transition to O/W microemulsion.

The change of the slope of log σ with increasing aqueous phasecontent is plotted in Fig. 3b. The abrupt change in d(logσ)/d(Φ) curvehas been used for indicating the occurrence of percolation behavior[34,35]. In Fig. 3b, the maximum of [d(logσ)/dΦ] is observed and the

Fig. 3. (a) Variations of conductivity (σ) of the empty (■) and β-carotene-loaded ( ) microemulsions as a function of aqueous phase content (Φ) along thedilution line L64 at 25 ± 0.1 ℃. Inset: Change of log (σ) with aqueous phasedilution along the same dilution line; (b) Plot of [d(logσ)/dΦ] as a function of Φfor both systems in (a).

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corresponding aqueous phase content is in line with Φp (as shown inFig. 3a), which confirms the presence of percolation behavior in thisregion. During the dilution range from 60wt% to 80wt% aqueousphase, the change of the slope of log (σ) is very little, which manifeststhe formation of B.C. structure in this dilution range, as has been re-ported by Feng et al. [33]. Additionally, the conductivity curves of bothsystems accord with each other basically, which signifies that β-car-otene incorporation does not affect the microstructural transitions.Thus, the results of conductivity measurement (as shown in Fig. 3)elucidate continuous microstructural transitions in the empty and β-carotene-loaded microemulsions upon aqueous phase dilution, from W/O micelles to B.C. type (between 60wt% ˜ 80wt% aqueous phase), andfinally to O/W structure (﹥80wt% aqueous phase).

3.4. Viscosity and microstructural transition

Viscosity measurements were carried out to obtain information oninternal structure changes in microemulsion systems. Microstructuralchanges of microemulsions can be attributed to either the change in theshape of the aggregates or the transition of microstructural types of themicroemulsion, wherein including droplet aggregation and fusion[36–38]. Fig. 4a depicts viscosity variations of β-carotene free andloaded microemulsions along the dilution line L64. All the measured

samples exhibit a Newtonian flow behavior. Bell-shaped viscosity pro-files of both systems can be observed, as reported in some nonionicmicroemulsions, Tween 60/R(+)-limonene/ ethanol/propylene glycol/water [39], Tween 80/butyl lactate/IPM/water microemulsion system[40] and so on. In Fig. 4a, the viscosity (η) increases slowly at the be-ginning of aqueous phase dilution, which is due to the formation ofdispersed water globules in continuous oil phase and the weak inter-actions between the droplets. With further dilution (Φ﹥20wt%), dis-persed water globules grow and the viscosity climbs sharply. The rapidincrease in viscosity is ascribed to the increasing droplet size andthereby enhanced attractive interactions and aggregations of swollenW/O droplets [26,40–42]. After reaching the maximum value of η (Φ=45wt% aqueous phase), further aqueous phase dilution leads to adecreasing viscosity.

Viscosity curves in Fig. 4a can be divided into three regions onmicroemulsion microstructure based on the first derivative of η as afunction of Φ ((dη/dΦ)˜Φ, see inset of Fig. 4a). The first transition pointin the plot of (dη/dΦ) vs Φ at 40 wt% aqueous phase may be related tothe structural transformation of microemulsion domains. After that, theminimum of dη/dΦ (at 60 wt% aqueous phase) occurs, which indicatesa formation of a bicontinuous structure. The similar identification onthe microstructural transition has also been presented in TX-100/oleicacid/water ternary system studied by Rahman et al. [36] and clove oil/water/2-propanol/Tween 20 system by Nazar et al. [37]. When Φ ishigher than 75 wt%, the values of (dη/dΦ) become constant, which canbe taken as an indication of further transition into O/W droplets fromthe B.C. structure. In this water-rich region, water becomes the con-tinuous phase and the viscosity slowly decreases due to the dilutioneffect of the O/W droplets [40]. Nevertheless, it has been frequentlyreported that a sharp decrease of viscosity after reaching its maximumreflects a progressive transition into O/W microemulsion [26,43]. Inthis work, we assume that the decrease of viscosity is attributed to theformation of a B.C. structure. It can be explained by the facts that this isa highly dynamic structure and the dynamics is enhanced owing to theincreasing amount of ethanol and glycerol present in surfactant inter-facial layer [7,44]. In a word, the viscosity results show continuoustransitions in microemulsion systems from swollen W/O micelles to abicontinuous structure (between 45wt% and 75wt % aqueous phase),with the system finally transforming into O/W microemulsion (Φ﹥75 wt%).

Furthermore, in Fig. 4a, similar trend can be observed on the visc-osity of empty and loaded microemulsions as a function of Φ. The in-corporation of β-carotene molecules in the microemulsions exhibits alittle higher viscosity than the empty ones. To reveal whether the in-crease of viscosity is due to the loaded β-carotene, we conduct furtherexperiments with increasing the amount of β-carotene in the con-centrate. Fig. 4b depicts the effect of β-carotene content variation onthe viscosity of microemulsions at 10˜75wt% aqueous phase dilution.The differences of viscosity between the empty and loaded systems aredistinct in the dilution range of 35˜50 wt% aqueous phase. That is, thehigher content of β-carotene is loaded, the larger viscosity the micro-emulsions will manifest, meaning that the solubilized β-carotene cansignificantly enhance the entanglement of surfactants hydrocarbontails. We thus conclude that the β-carotene molecules are mainly lo-cated in the palisade layer of the interfacial film of W/O microemulsion(35˜50 wt% aqueous phase). This conclusion is consistent with the re-sults in Fig. 2 that the IF values are greater than 1 at the same dilutionlevel. Once the bicontinuous phase is rich in water (Φ﹥50wt%), thedifferences in viscosity between the two systems are negligible, whichcan be concluded that the β-carotene molecules mostly move into theoil phase (reflected in the IF values less than 1; Fig. 2) and the sur-factant tails are not much influenced. We thus confirm that the reasonof significant decrease in SC as aqueous phase content exceeds 60wt%(as shown in Fig. 2) is mainly that, the curvature of interfacial film isgradually reduced resulting in fewer guest molecules embedded at theinterface [26]. However, a fact can’t be ignored that the IF value is

Fig. 4. (a) Viscosity of the empty (■) and β-carotene-loaded ( ) microemul-sions as a function of aqueous phase weight content (Φ) along the dilution lineL64 at 25 ± 0.1 ℃. Inset: The first derivative of the viscosity (dη/dΦ) as afunction of Φ. (b) Effect of β-carotene content in the concentrate on the visc-osity of the microemulsions.

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above 1 (Fig. 2) but the effect of β-carotene on the viscosity disappearsas the system is very diluted (Φ=90wt%). It can be deduced that someof the co-surfactants (EtOH and glycerol) gradually migrate out of theinterface when the microemulsion structure inverts into O/W type andtherefore the β-carotene molecules migrate into the surfactant inter-face. Similar interpretation has been supported by Kogan et al. [26].The cosurfactants desorption leaves more space for the β-carotene atthe interface, which is reflected in the IF value at 90 wt% aqueous phase(IF= 1.33; Fig. 2). The gradual decrease of viscosity upon aqueousphase dilution in the O/W region is owing to larger inter-droplet dis-tances and decreased interaction between the droplets [7,36,45],wherein the dilution effect is dominant and the effect of β-carotene onthe viscosity is minimal. Therefore, in addition to the dilution factor,the solubilization of β-carotene is related to the interfacial curvatureand the composition of the interface.

3.5. Microemulsion structure by SAXS

SAXS is a very powerful technique for the microstructural study ofmicroemulsions and here is used to identify structural differences inblank and β-carotene-loaded microemulsions. The scattering profiles ofthe two systems as a function of aqueous phase content are presented inFig. S2. The scattering profiles of all samples show a single peak atq≠0 followed by a high scattering angle tail, which are characteristicsof a typical microemulsion system [25,43]. With aqueous phase contentincreasing, scattering curve peaks of empty and β-carotene-loaded mi-croemulsions all shift to smaller q values, indicating an increase indroplet size in both systems [46] and the existence of β-carotene almosthas no influence on the size of domains in the microemulsions. At verylow aqueous phase content (Φ=15wt%), a broad scattering peak canbe observed, which is typical for the micellar system [47]. Values of theperiodicity (d) and correlation length (ξ) can be obtained by fitting thescattering curves to the Teubner-Strey equation [24] (Eq. (1)). Thevariations of the d and ξ parameters with increasing Φ are plotted inFig. 5.

Periodicity (d) reflects the distance between two opposite polarhead groups in the same droplet or the distances between two droplets,i.e., intermolecular distances [46]. The d values shown in Fig. 5a arederived from mean distances between particles for the investigatedmicroemulsion systems. In Fig. 5a, we find that the d values of bothsystems increase monotonically over the whole range of aqueous phasedilution. Moreover, in water-rich region (Φ﹥75wt %), the d values ofthe two systems increase steeply, which suggests larger distances be-tween the structural units in very diluted systems. This conclusioncorresponds well with the results generated through viscosity method.

With regard to correlation length (ξ), it is an useful parameter re-flecting the order degree of microemulsion system [43,46]. In Fig. 5b,typical bell-shaped plots can be observed and the ξ values of the loadedmicroemulsions are similar to those of the empty microemulsions. Thusit can be seen that the microstructural transformation occurs in bothsystems upon aqueous phase dilution and the β-carotene loaded doesnot have distinct effect on the degree of order in the system. Initially, ξincreases steeply with the increase in aqueous phase concentration. AsΦ﹥45wt%, ξ increases slowly and then reaches up to its maximum at55 wt% aqueous phase. Thereafter, the values of ξ remain approxi-mately constant up to 65 wt%. Beyond this value, ξ decreases linearly.The results can be interpreted as follows: when water acts as the dis-persed phase, increasing aqueous phase content will make the disperseddroplets swollen and lead to an increasingly ordered system withgrowth in ξ. While water is the continuous phase, addition of theaqueous phase will dilute the system and make the order degree de-crease (decline in ξ) [26,43]. In view of the above, the maximum valueof ξ is related to the formation of B.C. structures which exhibit a highdegree of order [47]. The results therefore confirm the continuoustransition of microstructure in the microemulsion systems, from W/Omicelles to more ordered bicontinuous phase, and finally to O/W

droplets. This identification result on microstructural transition isconsistent with that determined by conductivity and viscosity methods.

3.6. Surface tension and microstructural transition

Measurements of surface tension are carried out for the empty andβ-carotene-loaded microemulsions along the dilution line L64. The re-sults obtained from surface tension studies are depicted in Fig. 6. Thesurface tension initially decreases with increasing aqueous phase con-tent, but increases gradually upon dilution to 50wt%. Similar phe-nomena have been reported by Kaur et al. in Brij 96/butanol/eth-lyoleate/water microemuslion system [7]. The minimum of surfacetension at 50 wt% aqueous phase corresponds to the presence of a moreorganized microstructure [7,48], i.e. a bicontinuous structure, in bothmicroemuslion systems. It is noteworthy that the transition point cor-responds to that determined by viscosity and SAXS measurements. Afterpassing through the transition point, the surface tension increasesgently with dilution but increases linearly as Φ exceeds 70wt%. It canbe speculated that an increasing amount of water is on the outside ofthe oil droplets, i.e. the formation of O/W structure (Φ﹥70 wt%), whichresults in a regular increase in surface tension [48]. Additionally, in-corporation of β-carotene in microemulsion system doesn’t show mucheffect on surface tension but induces an obvious increment in the

Fig. 5. (a) The periodicity (d-values) and (b) correlation length (ξ-values) cal-culated from the SAXS measurements, along the dilution line L64 in.(■) emptyand ( ) β-carotene-loaded microemulsions at 25 ± 0.1 ℃.

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dilution range of 0˜20 wt% aqueous phase. This phenomenon suggeststhe possibility that the loaded β-carotene solubilizes in the continuousoil medium before the percolation behavior occurs (Φ=20wt%). Anincreasing force generated from the entanglement of β-carotene and theoil molecules operates on the surface of W/O microemulsion system,tending to shrink the surface and leading to a relatively high surfacetension. With further dilution, β-carotene locates mostly in the palisadelayer of the interfacial film or in the inner side of the dispersed phase,which has little influence on the surface tension of the microemulsionsystem. The results fit well with the calculated IF values in Fig. 2.

3.7. Microstructural transition by negative-stained TEM

The Tween 60/IPM/EtOH/glycerol/water microemulsion samplesand β-carotene loaded ones are imaged by negative-stained TEM. Thedimension of the system is clearly presented through TEM. From theimages in Fig. 7, still no obvious differences in particle size and shapecan be observed between the β-carotene free and loaded microemul-sions, which suggests that the incorporation of β-carotene has no no-ticeable effect on microstructure of the studied system. Along dilutionline L64, progressive transitions of microstructure occur in both sys-tems. The micrographs of microemulsion samples containing 30wt%aqueous phase significantly show spherical micelles (Fig. 7A1 and A2).

Then the bicontinuous structure is formed and accompanied with someintersecting filamentous aggregates, which may explain why the visc-osity of both microemulsions approaches to the maxima at ˜45 wt%aqueous phase (Fig. 4a). With further dilution, the filamentous ag-gregates in both systems disappear, while the bicontinuous structuresare completely formed (Fig. 7C1 and C2). Once the systems are muchdiluted (˜95wt%), spherical O/W structures with a diameter of 100 nmare ultimately formed in both systems (Fig. 7E1 and E2). In addition,the location and orientation of β-carotene molecules during micro-structural transitions of the system are schematically depicted in Fig. 8.

4. Conclusions

In this paper, we formulated a biocompatible microemulsion systemcomprising the pharmaceutically/cosmetically acceptable componentsTween 60/IPM/EtOH/glycerol/water. A large isotropic single-phaseregion was obtained in the pseudo-ternary phase diagram of the system.For consideration of applied research, we selected a dilution line toconstruct a U-type microemulsion as actives delivery system, whichprovided the advantage to form a β-carotene-loaded concentrate anddilute it progressively and fully by the aqueous phase. The SC of β-carotene in the studied microemulsion system drops upon dilution andthe locus of β-carotene are influenced by the microstructure of thesystem. As the solubility and bioavailability of the solubilizates aredirectly correlated to the microstructure of delivery vehicle [40,48], avariety of techniques were employed to characterize the structuraltransitions upon aqueous phase dilution, including conductivity, visc-osity, surface tension, SAXS, and TEM. From these experiments, it canbe concluded that the Tween 60/IPM/EtOH/glycerol/water systemundergoes a continuous transition from W/O micelles to an O/W mi-croemulsion (﹥85wt % aqueous phase), via a bicontinuous structure(˜50 wt% aqueous phase). Additionally, the structure of the system re-mains unaffected in the presence of β-carotene molecules. Before thepercolation behavior occurs (Φ﹤20 wt%), the β-carotene molecules aremainly solubilized in the continuous oil medium of the microemulsions,generating a high surface tension. Further addition of the aqueousphase (35﹤Φ﹤50wt%) makes the W/O micelles swollen and thereby theβ-carotene molecules gradually move into the interfacial layer, re-sulting in an increased viscosity of the loaded microemulsions. As theinterfacial curvature declines, the interconnected networks, i.e. B.C.structures, are formed and the β-carotene molecules gradually migrateinto the oil continuous phase, inducing the effect of β-carotene on theviscosity decreased. When Φ exceeds 85wt%, O/W structure is

Fig. 6. Variations of surface tension (γ) with weight fraction of aqueous phase(Φ) for the system of the empty (■) and β-carotene-loaded ( ) microemulsionsalong the dilution line L64 at 25 ± 0.1 ℃.

Fig. 7. TEM images of β-carotene-free (1) and β-carotene-loaded (2) microemulsions with aqueous phase dilution along the dilution line L64. A, B, C, D and Erepresent 30, 50, 75, 85 and 95wt% of aqueous phase, respectively.

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gradually formed and the β-carotene molecules are entrapped at theinterface once more because of the cosurfactants desorption in the in-terfacial layer. Furthermore, the SAXS measurements show a con-tinuous increase of structural dimensions upon dilution with aqueousphase and confirm the existence of more ordered bicontinuous struc-tures in the dilution range of 45˜75wt% aqueous phase. In future re-search, the formation of large bicontinuous region and O/W structureupon aqueous phase dilution will have important practical implicationsin water-insoluble nutraceuticals delivery applications. The corre-sponding β-carotene loaded formulations can also be developed as apotential drug/actives delivery system or biocompatible nano-templatefor other applications.

Notes

The authors declare no competing financial interest.

Acknowledgements

This research work was supported by the National Natural ScienceFoundation of China (No. 20573091) and Qing Lan Project of highereducation of Jiangsu Province ([2017] No. 5). The technical supportfrom the Testing Center of Yangzhou University is gratefully acknowl-edged.

Appendix A. Supplementary data

Supplementary material related to this article can be found, in theonline version, at doi:https://doi.org/10.1016/j.colsurfa.2019.01.041.

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