Structural transitions in cholesterol-based wormlike micelles induced by encapsulating alkyl ester oils with varying architecture

Journal of Colloid and Interface Science 378 (2012) 125–134

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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis

Structural transitions in cholesterol-based wormlike micelles induced by encapsulating alkyl ester oils with varying architecture Hala Afifi a, Göran Karlsson b, Richard K. Heenan c, Cécile A. Dreiss a,⇑ a

Institute of Pharmaceutical Science, King’s College London, 150 Stamford Street, London SE1 9NH, UK Department of Chemistry, BMC, Uppsala University, Box 759, Uppsala SE-751 23, Sweden c ISIS-CCLRC, Rutherford Appleton Laboratory, Chilton, Didcot OX11 0QX, UK b

a r t i c l e

i n f o

Article history: Received 14 February 2012 Accepted 5 April 2012 Available online 13 April 2012 Keywords: Wormlike micelles Polyoxyethylene cholesteryl ether Oil encapsulation Viscoelastic behaviour Small-angle neutron scattering Ring-like micelles

a b s t r a c t The effect of encapsulating oils on the phase behaviour and the microstructure of wormlike micelles formed by polyoxyethylene cholesteryl ether (ChEO10) and triethylene glycol monododecyl ether co-surfactant (C12EO3) was investigated using rheology, Cryo-TEM and small-angle neutron scattering measurements. Six alkyl ester oils bearing small, systematic variations in their molecular structure were encapsulated: ethyl butyrate (EB24), ethyl caproate (ECO26), ethyl caprylate (EC28), methyl enanthate (ME17), methyl caprylate (MC18) and butyl butyrate (BB44), where the subscripts refer to the length of the alkyl chain and fatty acid chain, respectively, on either sides of the ester link. The addition of alkyl ester oils to ChEO10/C12EO3 solutions promotes the longitudinal growth of the surfactant aggregates into wormlike micelles possessing an elliptical cross-section, with rminor 31 ± 2 Å and rmajor varying from 45 to 70 Å. At fixed alkyl chain length, oils with longer fatty acid chains were found to be more efficient in inducing wormlike micelle formation or their elongation, following the order: EC28 > ECO26 > EB24. Instead, at fixed fatty acid chain length, increasing the alkyl chain has a negative effect on the longitudinal micellar growth (MC18 > EC28 and EB24 > BB44). At high co-surfactant concentrations and in the presence of EB24, an unusual phase of ring-like micelles was detected. Overall, the orientation of the oil molecules within the micelles enables them to act as co-surfactants with a small head-group, decreasing the average cross-section area and promoting longitudinal growth of the micelles into worms. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Wormlike micelles are long, semi flexible aggregates which, above a system-dependent concentration, entangle into a transient network, imparting remarkable viscoelastic properties to its solutions. The earlier studies on wormlike micelles were largely focused on long chain cationic surfactants [1–3]. Later studies have shown that the growth of wormlike micelles can be promoted by the addition of co-surfactants or other low-molecular weight additives, such as short chain alcohols, counterions, salts and oppositely charged surfactants [4–6]. Recently, increasing interest has focused on the use of non-ionic, biocompatible and biodegradable surfactants, either for biomedical applications or because of environmental concerns. Polyoxyethylene cholesteryl ethers, sucrose esters, phytosterols and alkylglucosides are amongst the commonly reported environment-friendly surfactants forming wormlike micelles, usually in the presence of a co-surfactant [4,7–12]. This contribution focuses on wormlike micelles formed by mixing polyoxyethylene cholesteryl ether (ChEO10) with triethylene glycol ⇑ Corresponding author. E-mail address: [email protected] (C.A. Dreiss). 0021-9797/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcis.2012.04.014

monododecyl ether (C12EO3) and examines the effect of encapsulating oils with systematically varying molecular architecture on the phase behaviour. Wormlike micelle formation of polyoxyethylene cholesteryl ethers with either short EO-chain polyoxyethylene dodecyl ether [4,13], alkanolamides [14] or monoglycerides as cosurfactants [9,15], has been reported previously, starting with the work of Kunieda et al. [4]. It is well documented that the addition of oil affects the bulk behaviour and the microstructure of wormlike micelles [5,16– 18]. The phase behaviour however is highly dependent on the nature of the oil and the surfactant system [5,7,16,18–20]. The structural changes induced by oil addition usually fall under two main scenarios: the oil can either swell the micelles and induce a rod-to-sphere transition by increasing the natural curvature, or act as a co-surfactant by locating in the palisade layer and thus promote micellar growth. Understanding the effect of oil encapsulation into wormlike micelles is important to explore their use as delivery systems, as the oil could enhance drug encapsulation by increasing its solubilisation site, as well as significantly modify the bulk behaviour [15]. Recently, we reported studies on a viscoelastic micellar phase of wormlike micelles in mixtures of polyoxyethylene cholesteryl ether ChEO10 with lipophilic monoglycerides,

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Scheme 1. Chemical structure of polyoxyethylene cholesteryl ether (ChEO10) and triethylene glycol monododecyl ether (C12EO3).

Table 1 Chemical structure of the alkyl ester oils studied, their molecular weight (Mw) and log P values. The subscript represents the number of carbon atoms in the alkyl and the fatty acid chain of the oil, respectively. Oil Ethyl butyrate (EB24)

Chemical structure

H3C

O

H3C

O

H3C

O

CH3

Mw (g mol1)

Log P

116

1.8

144

2.8

172

3.8

144

2.8

158

3.4

144

2.8

O Ethyl caproate (ECO26)

CH3

O Ethyl caprylate (EC28)

CH3

O Methyl enanthate (ME17)

O CH3

H3C O Methyl caprylate (MC18)

O

CH3

H3C O Butyl butyrate (BB44)

H3C

O

CH3

O

at room temperature [15]. The encapsulation of pharmaceutical oils of different chemical structures (ethyl butyrate, ethyl caprylate, peppermint oil and tea tree oil) promoted the longitudinal growth of the micelles, while conserving the elliptical cross-section. Clear viscoelastic behaviour was observed with the addition of oils, even in the absence of co-surfactant, however at high cosurfactant concentration, the addition of oil reduced the viscoelasticity and phase separation was observed. In this contribution, our objective is to provide a rationale for the impact of oil architecture on the phase behaviour of wormlike micelles (Scheme 1), by using step-wise, systematic variations in alkyl ester oils chain length on either side of the ester link (Table 1). Rheological measurements are performed to monitor macroscopic changes, while small-angle neutron scattering and Cryo-TEM are employed to characterise concomitant microstructural changes. Six alkyl ester oils are used for this purpose: ethyl butyrate (EB24), ethyl caproate (ECO26), ethyl caprylate (EC28), methyl enanthate (ME17), methyl caprylate (MC18) and butyl butyrate (BB44) (Table 1). For ease of identification, each alkyl ester is abbreviated by two letters and a numerical subscript relating to the chain length. For instance, ethyl butyrate is abbreviated as EB24, where the first subscript gives the number of carbon atoms in the alkyl chain (ethyl group) and the second one in the fatty acid chain (butyrate group). Alkyl esters are used in the pharmaceutical field, in particular in transdermal formulations, for instance ethyl oleate, isopropyl myristate and isopropyl palmitate [21,22]. The alkyl ester oils studied

here are mainly used as aroma compounds [23–25], however ethyl butyrate and ethyl caprylate have also been studied to encapsulate hydrophobic drugs [26]. 2. Experimental section 2.1. Materials Polyoxyethylene cholesteryl ether (ChEO10) was purchased from Ikeda Corporation, Yokohama, Japan. Triethylene glycol monododecyl ether co-surfactant (C12EO3) (>98%), ethyl butyrate (EB24) (99%), ethyl caprylate (EC28) (99%), ethyl caproate (ECO26) (99%), methyl caprylate (MC18) (99%), methyl enanthate (ME17) (99%) and butyl butyrate (BB44) (98%) were all obtained from Aldrich Chemical Co. Ltd., UK. All chemicals were used as received unless otherwise stated. Deuterium oxide (99.9 atom % D) was purchased from Aldrich Chemical Co. Ltd., UK. 2.2. Rheological measurements Rheological measurements were performed on a dynamic strain-controlled rheometer (ARES, TA instruments) using coneand-plate geometry, with a temperature-controlling Peltier unit and a sample cover to minimise evaporation. Two types of rheological measurements were performed: oscillatory-shear measurements (frequency sweep tests performed in

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the linear viscoelastic regime, as determined by dynamic strain sweep measurements) and steady shear-rate viscosity measurements. Measurements were carried out in duplicates or triplicates for each sample, with very good reproducibility. The results reported in this manuscript are examples of typical data obtained, not averages. Samples for rheological measurements were vortex-mixed and kept in a water bath at 25 °C for 24 h to ensure equilibration before performing the measurements. Zero-shear viscosity (g0) was obtained manually from the flow curves. The plateau modulus (G0) is taken as the value of G0 at x = 100 rad s1 and the relaxation time (sR) is estimated as sR = 1/xc, where xc is the oscillation frequency at which G0 = G00 (cross-over), where G0 and G00 are the storage and loss modulus, respectively. 2.3. Small-angle neutron scattering (SANS) SANS measurements were performed on LOQ instrument at the ISIS pulsed neutron source (ISIS, Rutherford-Appleton Laboratory, STFC, Didcot, Oxford) and V4 instrument at the Hahn-MeitnerInstitute (now HZB), Berlin, Germany. On LOQ, the neutron scattering pattern of the samples is collected on a two-dimensional detector placed at 4.1 m from the sample and the scattering vector q covers a range from 0.009 to 0.29 Å1, using the time-of-flight technique with neutrons of wavelength 2.2–10 Å. V4 covers a q range from 0.004 to 0.37 Å1, using a wavelength of 10 Å and three detector distances of 1, 4 and 12 m from the sample. The samples were prepared in D2O to optimise the contrast with the protonated surfactants and oils. They were placed in clean disc-shaped quartz cells (Hellma) of 1 or 2 mm path length and the measurements were carried out at 25 °C. Raw data were corrected for detector efficiency, transmission, scattering from the empty cell, pure D2O and background radiation. The data were then converted to the differential scattering cross-sections (in absolute units of cm1) using the standard procedures at ISIS [27] and HZB. 2.4. SANS data fitting Data presenting a typical Kratky behaviour in a [q2I(q) vs q] representation (‘bell-shape’ scattering curve with a linear slope at low q) were tested against the Kratky–Porod worm-like chain model, which describes the scattering from elongated objects characterised by their local rigidity [28,29] (see Supplementary data for a description of the model). SANS data were fitted using a model for cylinders with an elliptical cross-section, using routines from the NIST SANS group encoded within SansView programme from the DANSE project [30]. The expression of the intensity, where L is the length of the cylinder and rminor and rmajor are the minor and major radii, respectively, is the following:

IðqÞ ¼

Scale V cyl

Z

dW

Z

d/

Z

pðh; /; WÞF 2 ðq; a; WÞ sin hdh þ bkg

ð1Þ

with the functions:

Fðq; a; WÞ ¼ 2

J 1 ðaÞ sinðbÞ  a b 2

a ¼ q  sinðaÞ½r2major sin W þ r 2min or cos2 W1=2 b¼q

L cosðaÞ 2

ð2Þ ð3Þ

Table 2 Mass density values and calculated scattering length densities of the surfactants, oils and D2O. Chemical

Density (g/mL)

Scattering length density (Å2)

D2O ChEO10 C12EO3 EB24 EC28 ECO26 BB44 MC18 ME17

1.107 1.065 0.927 0.878 0.867 0.869 0.869 0.877 0.870

6.4  106 4.2  107 1.3  108 3.0  107 10.0  108 1.8  107 1.8  107 1.4  107 1.8  107

parameter was left to float and then the value returned by the fit checked against its calculated value to confirm consistency of the fit (an error of ±10% was deemed acceptable). A core/shell ellipsoidal cylinder model was also employed to fit the data (where the scattering length density of surfactant tails and heads are considered separately rather than taking their volume-weighted average). Good fits with similar parameters were obtained, however the fits were not sensitive to the position of the oil when different scenarios were tested. Therefore, we opted for the hard ellipsoidal cylinder model for simplicity and to avoid making arbitrary assumptions about the localisation of the different species. Models for hollow cylinders (tubes) and vesicles were also used to fit some SANS curves using the FISH programme [27]. The model is described in Supplementary data. Fitted curves included numerical smearing to approximate the instrumental q resolution and also a flat background to allow for residual incoherent scattering not present in the solvent background cell subtraction. The scattering length densities and the mass density of the surfactants and oils used are given in Table 2. 2.5. Cryogenic Transmission Electron Microscopy (Cryo-TEM) Cryo-TEM measurements were performed with a Zeiss TEM 902A instrument (Carl Zeiss NTS, Oberkochen, Germany). The instrument was operated at 80 kV in zero-loss bright-field mode. Digital images were recorded under low dose conditions with a BioVision Pro-SM Slow Scan CCD camera system Proscan elektronische Systeme GmbH, Germany and iTEM software (Olympus Soft Imaging Solutions GmbH, Münster, Germany). An underfocus of 1–3 lm was used in order to enhance contrast. The preparation procedure has been described in detail elsewhere [6]. Specimens for examination were prepared in a climate chamber with temperature and humidity control (Temperature 25 °C and relative humidity of approximately 98–99 %). Thin films of sample solution were prepared by placing a small drop of the sample on a copper grid supported perforated polymer film, covered with thin carbon layers on both sides. After the drop was blotted with filter paper, thin sample films (10–500 nm) spanned the holes in the polymer film. Immediately after blotting, the samples were vitrified by plunging them into liquid ethane, held just above its freezing point. Samples were kept below 165 °C and protected against atmospheric conditions during both transfer to the TEM and examination. Several images were taken of each sample studied, which were all consistent, and representative examples are presented here. 3. Results

ð4Þ

where the ‘scale’ is a scaling factor containing information such as volume fraction and contrast, the angles h and / define the orientation of the axis of the cylinder and the angle w is the orientation of the major axis of the ellipse with respect to the vector q. The ‘scale’

3.1. Pure ChEO10/C12EO3 wormlike micelles The rheological behaviour of ChEO10/C12EO3 micellar solutions is used as a reference for comparison with the samples containing oil (in the following section). Results on the ‘naked’ micelles have

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H. Afifi et al. / Journal of Colloid and Interface Science 378 (2012) 125–134 Table 3 Rheological parameters of ChEO10/C12EO3 solutions at 10 wt.% ChEO10 and 0–1.5 wt.% C12EO3 in the absence and presence of alkyl ester oils. Cross-section radii (rmajor) and apparent lengths (L) obtained from the elliptical cylinder model of 10-fold dilution of ChEO10/C12EO3 solutions at 10 wt.% ChEO10 and 0–1.5 wt.% C12EO3 in the absence and presence of alkyl ester oils. rminor is 31 ± 2 Å for all samples. Parameters in bold font correspond to hazy/turbid solutions.

Zero-shear viscosity (Pa.s)

1000 100 10

1 0.1

C12EO3 (wt.%)

Additives

G0 (Pa)

g0 (Pa s)

sR (s)

rmajor (Å)

L (Å)

0

No oil 1%EB24 1%EC28 1%ECO26 1%ME17 1%MC18 1%BB44 2%EB24

ND ND 7 10 13 14 ND 3

0.002 0.004 6 3 1 11 0.006 3

ND ND 0.8 0.25 ND 0.6 – <0.01

70 58 58 56 51 53 53 50

155 155 300 260 450 500 220 157

0.5

No oil 1%EB24 1%EC28 1%ECO26 1%ME17 1%MC18 1%BB44 2%EB24

ND 28 14 21 20 21 2 16

0.03 25 11 305 710 550 0.2 480

– 1.6 >10 >10 >10 >10 – >10

51 50 50 54 51 51 51 48

280 300 670 650 700 720 350 300

1.0

No oil 1%EB24 1%EC28 1%ECO26 1%ME17 1%MC18 1%BB44 2%EB24

16 37 10 17 43 46 12 16

10 562 8 220 35 56 450 262

0.6 >10 0.8 >10 0.6 1.25 >10 2.5

51 45 50 51 51 – 48 45

400 500 670 650 730 – 600 500

1.5

No oil 1%EB24 1%EC28 1%ECO26 1%ME17 1%MC18 1%BB44 2%EB24

23 27 3 6 22 4 31 ND

410 159 2 58 11 8 109 3

>10 >10 >1 >6 ND 4 6.2 –

48 45 – 51 – – 48 –

430 600 – 650 – – 600 –

0.01

0.001 0

0.5

1

1.5

2

2.5

3

%C12EO3 Fig. 1. Zero-shear viscosity of 10 wt.% ChEO10 solutions as a function of C12EO3 concentration, with no oil ( ) and 1 wt.% of the following oils: EB24 ( ), EC28 ( ), ECO26 ( ), ME17 (N), MC18 ( ), BB44 (s) and 2 wt.% EB24 ( ), with 0 to 3 wt.% C12EO3.

been reported before; for this reason, they are not discussed in detail here, and we refer the reader to previous papers [4,31] and to Figs. 1 and 2 in Supplementary data. Steady-shear and oscillatory-shear measurements reveal a viscoelastic behaviour in 10 wt.% ChEO10 solutions with 0.5 to 2.5 wt.% C12EO3 (Supplementary data, Figs. 1 and 2). In particular, the predominantly solid-like viscoelastic behaviour (G0 > G00 ) obtained with 1–2.5 wt.% C12EO3 (Supplementary data, Fig. 2) and the significant increase in zero-shear viscosity observed up to 2 wt.% C12EO3 (Supplementary data, Fig. 1) have been attributed to the formation of wormlike micelles and their entanglement into a transient network [4,31]. Co-surfactant concentrations above 2 wt.% lead to a decrease in zero-shear viscosity, a behaviour widely reported for wormlike micelles and commonly attributed to chain branching (Fig. 1) [32–34], where the formation of junction points, which are able to slide along the worms length, act as a mechanism to release the stress. Above 3 wt.% C12EO3, turbid solutions were obtained. The behaviour obtained for viscoelastic ChEO10/C12EO3 solutions (Supplementary data, Fig. 2) cannot be described by the Maxwell model. This model, although commonly found to describe wormlike micelles response, is not universal (e.g. Ref. [35]). We note in particular for some samples (e.g. 2% C12EO3, Supplementary data, Fig. 2), that no visible cross-over point for G0 and G00 is detected in the frequency range investigated, making these samples particularly solid-like. SANS measurements were performed on these samples [31], which confirmed the presence of rod-like micelles [13,31], however with an unusual ellipsoidal cross-section (with rminor = 33 Å and rmajor = 70 Å, Table 3), as also previously reported with monoglycerides as co-surfactants [15]. The decrease in the cross-section radius induced by the intercalation of the co-surfactant smaller head-groups and the increase in the apparent length (Table 3), both confirm the axial growth of the micelles into worms. CryoTEM images (published elsewhere [31], and Supplementary data, Fig. 3, with 1 wt.% C12EO3) confirm the presence of wormlike micelles with different widths and contrast, thus pointing to oval cross-sections or ribbon-like structures. The elongated sectional area of ribbons causes more or less electron scattering depending on its orientation with respect to the direction of electron radiation; this appears in the 2D images as if the threadlike structures have different thicknesses along their length. At higher co-surfactant concentrations, samples become turbid (with 3 wt.% C12EO3)

Elliptical-cylinder model

ND: Not detectable.

and the presence of more than one phase is detected in CryoTEM images (data not shown), which correspond to the descending part of the zero-shear viscosity curve (Fig. 1). 3.2. Encapsulation of alkyl ester oils in the micelles 1 wt.% (and 2 wt.%, only for EB24) of six alkyl ester oils: ethyl butyrate (EB24), ethyl caprylate (EC28), ethyl caproate (ECO26), methyl caprylate (MC18), methyl enanthate (ME17) and butyl butyrate (BB44), were solubilised in 10 wt.% ChEO10 solutions with increasing amount of C12EO3 (from 0 to 3 wt.%). The alkyl esters have varying alkyl and fatty acid chain lengths (Table 1). The data are interpreted and discussed in terms of: (1) the fatty acid chain length for a fixed alkyl chain (EB24 vs ECO26 vs EC28 and ME 17 vs MC18), (2) the alkyl chain length for a fixed fatty acid chain (EB24 vs BB44 and EC28 vs MC18) and (3) the position of the ester group for a fixed total number of carbon atoms (BB44 vs ECO26 vs ME17). 3.2.1. A. Rheological measurements 3.2.1.1. Steady-shear viscosity. Fig. 1 shows the change in zeroshear viscosity as a function of C12EO3 concentration in the presence and absence of the various oils. In the absence of co-surfactant (0% C12EO3), an increase in the zero-shear viscosity is observed with the addition of alkyl esters, except with EB24 (1%)

H. Afifi et al. / Journal of Colloid and Interface Science 378 (2012) 125–134

and BB44, which have the shortest of all fatty acid chains studied (4C). This increase is more remarkable with EC28 and MC18, which have the longest fatty acid chain (8C), where g0 increases by three and four orders of magnitude, respectively. With 0.5 and 1 wt.% co-surfactant, the addition of 1% oil leads to a substantial increase in viscosity, with the exception of the system containing EC28 (Table 3). In the case of EC28, which has the longest chain (10C), g0 does not increase beyond the initial (high) value. This probably reflects the turbidity of EC28 solutions when in the presence of C12EO3 (Table 3), suggesting either phase separation or the formation of structures other than wormlike micelles, such as large lamellar aggregates. Apart from EC28, all alkyl esters show the same pattern, with g0 going through a maximum followed by a gradual decrease with increasing amount of C12EO3 (at fixed oil concentration). However, this maximum in g0 is shifted to lower co-surfactant concentration: 0.5 wt.% C12EO3 with ME17, MC18, ECO26 and 1 wt.% with EB24 and BB44, instead of 2 wt.% co-surfactant in the absence of oil. Therefore, the solubilisation of a small amount of oil could be promoting the formation of wormlike micelles (higher initial g0 values), and subsequently their branching (g0 peak position), at lower co-surfactant concentration. By increasing the amount of encapsulated EB24 to 2 wt.%, a shift in the g0 peak to a lower concentration of C12EO3, compared to 1 wt.% EB24, is observed (from 1 to 0.5 wt.%), suggesting that increasing the oil content could have a positive effect on inducing wormlike micelle formation. Overall, it is clear that oils with longer fatty acid chains (ME17, MC18, ECO26) shift the g0 peak and lead to higher values of g0 than in the absence of oil and than oils with shorter fatty acid chains (EB24 and BB44). However, beyond a critical molecule size phase separation occurs, as is seen with EC28, which has the longest fatty acid chain and larger Mw and log P value (partition coefficient between an organic – octanol – and aqueous phases) (Table 1). 3.2.1.2. Oscillatory-shear measurements. Fig. 2 shows the variation of G0 and G00 as a function of shear frequency in 10 wt.% ChEO10 aqueous solutions with 0–1.5 wt.% C12EO3 in the absence and presence of oil. To improve clarity, the frequency sweep curves are presented in two groups: EB24, EC28 and ECO26 in the left hand-side figures (a, c, e) and MC18, ME17, BB44 and no oil in the right hand-side figures (b, d, f). The results are discussed as a function of increasing C12EO3 concentration. In the absence of co-surfactant (Fig. 2 a and b), a clear solid-like viscoelastic behaviour (G0 > G00 ) is observed over some region of the frequency spectrum with alkyl esters of longer fatty acid chains: EC28, ECO26, MC18 and ME17; within that group, the shortest relaxation time (sR, taken as the cross-over between G0 and G00 ) is obtained with ME17 (Table 3). It is the smallest oil amongst these four (Table 1), differing from MC18 only by one carbon in the fatty acid chain. MC18 instead has a rheological behaviour very similar to EC28 (Table 3), thus again pointing to a critical role of the fatty acid chain length. This assumption is also supported by the observation that the solutions containing the two oils with the shortest fatty acid chain, EB24 and BB44 (Fig. 2a and b), exhibit a liquid-like behaviour, similar to the one observed in the absence of oil (Fig. 2b), however with slightly higher shear moduli values (Table 3). The remarkable change in the rheological response obtained by adding either EC28, ECO26, MC18 or ME17 to pure ChEO10 micelles must reflect drastic structural changes induced by oil solubilisation. In the presence of a small amount of C12EO3, namely, 0.5 wt.% (Fig. 2c and d), a clear solid-like behaviour is obtained with all but one of the alkyl esters (BB44), with very similar values of the moduli, while liquid-like behaviour is observed in the absence of oil. Very long relaxation times (which fall outside the frequency

129

range measured, i.e. higher than ca. 10 s) are observed with the long fatty acid chain oils, EC28, ECO26, MC18, ME17, and with 2 wt.% EB24, while with only 1 wt.% EB24 a short sR of 1.6 s is obtained (Table 3). By increasing the concentration of C12EO3 to 1 wt.%, a solid-like behaviour is observed in the absence and presence of all oils (including BB44) (data not shown), with the longest relaxation times obtained with EB24, ECO26 and BB44, while a decrease in sR is seen with alkyl esters of longer fatty acid chain, namely EC28, ME17 and MC18. Concomitant to this decrease in relaxation time, MC18 and ME17 also show a decrease in g0 and surprisingly, an increase in G0 (Table 3). The viscosity and the relaxation time are related by [36]:

g0 ¼ G0  sR

ð5Þ

And the theory of transient networks predicts [36]:

G0 ¼ v eff  R  T

ð6Þ

where R is the universal gas constant, T is the temperature and veff is the number of effective or active elastic chains, related to the entanglement density. The decrease in the relaxation time with MC18 and ME17 above 0.5 wt.% C12EO3 suggests a faster relaxation mechanism than chain breaking and reptation, probably chain branching, which is accompanied with a decrease in the zero-shear viscosity (Table 3) [4,32,37,38]. G0 is proportional to the density of cross-links between the chains (Eq. (6)); therefore, the increase in G0 obtained with MC18 and ME17 above 0.5 wt.% C12EO3, accompanied by the decrease in both sR and g0, points to the increase in the number of connection points resulting from branching [7,39]. The addition of oil at co-surfactant concentration of 1.5 wt.% (Fig. 2e and f) is accompanied by a decrease in the rheological parameters (compared to the ‘naked’ wormlike micelles), although still predominantly solid-like, except for 2 wt.% EB24, the smallest oil, which leads to a liquid-like viscoelastic response. In summary, the results of both steady-shear and oscillatoryshear measurements therefore show that the addition of a small amount of oil in the absence of co-surfactant leads to a more solid-like viscoelastic behaviour and higher viscosity values with EC28, ECO26, MC18 and ME17; a behaviour only seen at a low co-surfactant concentration (0.5 wt.%) in the case of EB24. Overall, if we compare the rheological parameters in the presence of oil at their respective zero-shear viscosity peak, very similar values are obtained, reflecting the fact that the differences in behaviour between the oils is simply a ‘shift’ to lower co-surfactant concentration (Table 3). At higher concentrations of co-surfactant (for e.g. at 2 wt.% C12EO3), where an entangled network of wormlike micelles is present ([4,31] and Supplementary data, Fig. 2), the addition of oil results in a decrease of the elasticity with the smaller oils (1 wt.% EB24 and BB44, Table 3) and leads to turbid solutions with the larger ones (EC28, ECO26, MC18, ME17 and 2 wt.% EB24, Table 3), below the concentration where it occurs in the absence of oil. The length of the fatty acid chain does seem to play a key-role in the onset of solid-like behaviour, as measured by the value of the plateau modulus and the height and position of the zero-shear viscosity peak. These results are discussed in more detail in the next section, in the light of the microstructural studies by SANS and Cryo-TEM. 3.2.2. B. Structural study: SANS and Cryo-TEM SANS measurements were performed on 10-fold diluted solutions of the samples to avoid interference effects, which would make data analysis very difficult and modelling ambiguous. Although the contour length and persistence length may be affected by concentration, trends in the shape parameters are expected to be conserved, and therefore comparisons between different samples are still valid.

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H. Afifi et al. / Journal of Colloid and Interface Science 378 (2012) 125–134

Fig. 3 shows the scattering intensity as a function of the scattering vector from 10-fold dilutions of 10 wt.% ChEO10 with 0–1 wt.%

a

C12EO3 solutions in the absence and presence of alkyl esters. In the absence of co-surfactant (Fig. 3a), no change in the general shape of

b

100

100

1%EB24 1%EC28

No oil

0% C12 EO3

1% ME17

1%ECO26

10

G' G'' (Pa)

G' G'' (Pa)

10

1

0.1

1

10

0.01 0.1

100

Angular frequency (rad.s-1)

1

10

100

Angular frequency (rad.s-1)

d

100

100

10

10

G' G'' (Pa)

G' G'' (Pa)

1

0.1

0.01 0.1

c

0% C12 EO3

1% MC18

1

0.1

1

0.1

1% EB24

No oil

2% EB24

1% ME17

1% EC28 1% MC18

1%ECO26

0.01 0.1

e

1

10

0.01 0.1

100

Angular frequency (rad.s-1)

f

100

1

10

100

Angular frequency (rad.s-1) 100

1% EB24 2% EB24

No oil

1.5% C12EO 3

1% MC18

1% ECO26

1.5% C12 EO3

1% BB44

10

G' G'' (Pa)

G' G'' (Pa)

10

1

1

0.1

0.1 0.1

1

10

Angular frequency (rad.s-1)

100

0.1

1

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Fig. 2. Variation of the storage modulus G0 (open symbols) and loss modulus G00 (filled symbols) as a function of the oscillatory shear frequency in solutions of 10 wt.% ChEO10 without oil ( ) and in the presence of 1 wt.% of the following oils: EB24 ( ), EC28 ( ), ECO26 ( ), ME17 (N), MC18 ( ), BB44 (s) and 2 wt.% EB24 ( ), with (a and b) 0, (c and d) 0.5 and (e and f) 1.5 wt.% C12EO3. Symbols match the ones used in Fig. 1 for easier readability. Oils showing liquid-like behaviour (G00 > G0 ) at specific co-surfactant concentration with very low values of G0 and G00 were removed from the figure for better visibility (2 wt.% EB (2a), BB (2b and d), EC (2e) and ME (2f)).

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Fig. 3. SANS experimental curves of 10-fold dilutions of 10 wt.% ChEO10 micellar solutions with 0, 0.5 and 1 wt.% C12EO3 (a, b and c, respectively) in the absence and presence of alkyl ester oils in D2O. The curves are staggered by multiples of 2 with respect to each other for clarity purposes. Fits to the elliptical cylinder model are represented by solid lines. d. SANS experimental curves from a 10-fold dilution of a 10 wt.% ChEO10 and 1.5 wt.% C12EO3 solution in the presence of 1 wt.% MC18 in D2O. Fit to a vesicle (h) and a hollow cylinder ( ) models are represented by a solid line. The data for the hollow cylinder were scaled up by 5 for clarity. Matching symbols to Figs. 1 and 2 have been used for better readability.

the scattering pattern is detected with the addition of EB24 (1 and 2 wt.%) and BB44: the scattering curve shows a near-plateau at very low-q, suggesting an object of finite dimensions, in good agreement with the rheology data, which show a conservation of the predominantly liquid-like behaviour (Fig. 2a and b). Instead, the addition of the other oils leads to an increase in the intensity and a q1 dependence, the signature of scattering from rod-like structures, as well as the disappearance of a low-q plateau, thus suggesting very elongated objects. This correlates with the increase in solid-like response observed in the oscillator measurements (Fig. 2). The increase in the scattering intensity follows the order MC18 > ME17 > EC28 > ECO26 (Figs. 3a–c). A Kratky–Porod representation of the data for samples containing 1 wt.% MC18, ME17, ECO26 and EC28 in the absence of co-surfactant, gives a linear plot over a wide q-range (Supplementary data, Fig. 4), confirming the presence of elongated cylindrical micelles of cross-section radii ca. 45–47 (±2 Å) and mass per unit length ML = 2.5 ± 0.5 kg mol1 Å1, a value comparable to the one obtained for ChEO10 pure micelles (ML = 3.0 ± 0.5 kg mol1 Å1 [31]). Good fits to the elliptical cylinder model were obtained (Fig. 3a– c, solid lines) and values of the cross-section radii obtained from the model are shown in Table 3. It was not possible to obtain good fits with rods of circular cross-section – even by incorporating instrumental smearing – as the lines of fit deviated from the data

at high q, as was reported previously with monoglycerides instead of C12EO3 as co-surfactants [15]. This particular shape must therefore result from the packing constraints imposed by the bulky, rigid cholesterol moieties into the core. In the presence of 0.5 wt.% C12EO3 (Fig. 3b), a similar scattering pattern (q1 behaviour, reflecting rods), is obtained with all oils, with the exception of BB44, which again correlates well with the liquid-like behaviour observed with this particular oil (Fig. 2d, Table 3). With a further increase in C12EO3 concentration to 1 wt.%, an increase in the scattering intensity and q1 behaviour are observed both in the absence and presence of all oils, with the exception of the system containing MC18 (Fig. 3c), where a small bump, which is more prominent at 1.5 wt.% C12EO3 (Fig. 3d), is detected at intermediate q. A reasonable fit to a ‘tube’ model (cf Supplementary data for the description of the model) was obtained for this particular sample, with a radius of 97 Å and a core radius size of 35 Å, thus of 62 Å thickness, a length that corresponds approximately to 2 ChEO10 molecules [15]. The length of the tube is 110 Å, which is rather short, probably reflecting rods flatteningup to sheets (lamellar structures) (Fig. 3d). An equally satisfactory fit was obtained by using a vesicle model of ca. 91 Å radius and bilayer thickness of 42 Å (Fig. 3d). It is not possible to distinguish unambiguously between these two structures and attempting to

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refine the fits would be quite vain given the likely presence of a mixture of various types of aggregates and the need to make a number of assumptions. Both fits however suggest the presence of lamellar structures, which would account for the turbidity of the samples. The major cross-section radii (for all samples rminor = 31 ± 2 Å) and the apparent lengths obtained both from the fits to the elliptical cylinder model for ChEO10/C12EO3 solutions are reported in Table 3, both in the absence and presence of the oils. At fixed C12EO3 concentration, a decrease in micellar radius rmajor (compared to the original short ChEO10 rods cross-section) is observed with the addition of oil, and an increase in micellar length with the longer chain oils (and at high C12EO3 concentration also with the shorter oils). Unlike long chain hydrocarbon oils [40], the alkyl ester oils studied here do not swell the core (which would give an increase in cross-sectional radius, and probably a rod-to-sphere transition) but mix with ChEO10, acting as co-surfactants, and thus leading to a decrease in the average cross-section of the aggregates, due to the smaller size of the oil molecules. Apart from the specific sample mentioned above (1.5 wt.% C12EO3 and MC18, Fig. 3d), all scattering data show elongated wormlike micelle structures, even beyond the zero-shear viscosity peak (except with EC28), thus for these samples, it appears that the decrease in zero-shear viscosity can be attributed to the branching of the worms (which is not detected in a SANS experiment), in agreement with the increase in G0 and decrease in sR (see Table 3 and discussion above). We report however below Cryo-TEM pictures of a turbid sample with predominantly liquid-like behaviour (10 wt.% ChEO10/1 wt.% EB24/3 wt.% C12EO3, Fig. 4b) showing an unusual micellar phase, which points out to the possibility of other structures than the widely invoked ‘lamellar aggregates’ for turbid samples.

Fig. 4. Cryo-TEM images of 10 wt.% ChEO10 with 1 wt.% EB24 and a. 1 wt.% and b. 3 wt.% C12EO3. The black and white arrow heads (4a, enlarged section) point to wormlike micelles with different thicknesses and the black arrows to ring-like micelles and wormlike micelles in the process of folding into a ring (4b, enlarged section). The scale bars represent 100 nm.

The worms here were not sufficiently short, compared to the qrange available, to use a flexible rod model. In this model, the reported values of ‘length’ are likely to correspond to a persistence length, as the rheology and microscopy (see below) suggest far longer total contour lengths. We note that, for the longer rods, a wide range of cylinder lengths give almost equally good fits (visually) to the SANS data. The values shown in Table 3 (which were obtained by letting both radii and length float) correspond to the best least square fits. Cryo-TEM images provide visual evidence of wormlike micelle formation in the presence of oil. Fig. 4 shows images of 10 wt.% ChEO10 solutions with 1 wt.% EB24 at 1 wt.% C12EO3 (corresponding to the zero-shear viscosity peak for this particular oil, Fig. 1) and 3 wt.% C12EO3 (beyond the viscosity peak, cf Fig. 1), which was selected to bring more insight into the composition of turbid samples (often attributed to lamellar aggregates, but rarely assessed). For the solutions containing 1 wt.% C12EO3 and 1 wt.% oil (Fig. 4 a), long wormlike micelles with an elliptical cross-section (ca. 35 ± 5 Å) can be seen, in agreement with both the SANS results (Fig. 3, Table 3) and the solid-like behaviour observed in the rheology measurements (Fig. 2). This unusual elliptical cross-section can be attributed to the surfactant structure, where the rigid cholesterol groups tend to form long rods [41], while the large polar PEO head-groups support a stronger curvature. A similar ‘structural compromise’ has been reported with the same surfactant (ChEO10) and a different co-surfactant [15] and with micelles formed by fluorinated cationic surfactants [42]. Interestingly, with 3 wt.% C12EO3 (Fig. 4b), at least two phases are detected: a few worms, as well as spherical particles, reminiscent of vesicles, with a radius of ca. 80 Å (black arrows). These structures however are much smaller than pure C12EO3 vesicles [43] and anyway extremely small to be vesicles. They are unlikely to be loops of wormlike micelles, as they are of extremely regular size and should be visible in different positions, which is not the case here. Instead, these structures are most likely to be ring-like micelles; they have in particular a very similar thickness to that of the worms (incompatible with a bilayer structure). In Fig. 4b, the presence of a number of short worms is detected, which could therefore fold into rings; in addition, the presence of some incomplete rings (Fig. 4b, enlarged section) confirm that the wormlike micelles are breaking-up into ring-like micelles in the presence of oil and at high co-surfactant concentration. We note also that in the regions where they are detected, the ring-like micelles are arranged in a very regular pattern, showing hexagonal close-packing. The presence of ring-like micelles may account for the liquidlike behaviour and the very low viscosity value (0.5 Pa s) of the solutions (data not shown), rather than the lamellar structures or branching usually invoked in the descending part of the zero-shear viscosity [4,37,38]. The corresponding SANS curve (10 wt.% ChEO10/3 wt.% C12EO3/ 1 wt.% EB24, Supplementary data, Fig. 5) shows a near q2 behaviour, thus departing from elongated rod shapes, and could not be fitted with the elliptical cylinder model; it is clear from the CryoTEM picture that this sample comprises a mixture of structures, and therefore we did not attempt further to fit it. These ring-like micelles are quite intriguing and have not been reported extensively. Examples in the literature include ring-like micelles in dimeric surfactants [44] and saponin/cholesterol mixtures [45,46], where similar patterns were obtained from TEM measurements [45]. In saponin/cholesterol mixtures [45], wormlike micelles were clearly shown to act as precursors – as also observed in this work – by first folding, followed by ring-like micelles budding off [45]. The particular structure of cholesterol and the packing constraints generated by this rigid hydrophobic moiety may be a key to this unusual micellar phase.

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4. Discussion The combination of techniques used for the characterisation of the micellar solutions, both from a macroscopic (rheology) and structural viewpoint, all point to the formation of wormlike micelles at lower co-surfactant concentration in the presence of oil. The encapsulation of the longer fatty acid-chain oils, ECO26, EC28, ME17 and MC18, induces the uni-dimensional growth of ChEO10 micelles into worms in the absence of the co-surfactant C12EO3 (Figs. 1 and 2a and b and 3a). The effect of oil solubilisation on the geometry of the aggregates can be explained in terms of the packing of the oil into the micelles, which is determined by its chemical structure. Alkyl esters are oils of moderate hydrophobicity (Table 1). They contain a polar group (the ester moiety) and are therefore likely to orientate within the aggregates with this polar group at the shell/core interface and their hydrophobic group (alkyl chains) buried inside the core of the aggregates, thus promoting rod elongation by acting as co-surfactants (decreasing head-group area and thus decreasing the aggregates curvature). The differences in behaviour are discussed and rationalised from three different viewpoints: (1) the length of the fatty acid chain, (2) the length of the alkyl chain and (3) the position of the ester group for a fixed number of carbon atoms (i.e. the relative importance of fatty acid vs alkyl chain). EB24, ECO26 and EC28 have an ethyl group as the alkyl chain and differ in the fatty acid chain length: C4, C6 and C8, respectively. EB24 was seen to be less efficient in driving wormlike micelle formation (elongation took place at 0.5 wt.% C12EO3), compared to ECO26 and EC28 (elongation took place in the absence of co-surfactant), as assessed from the onset of solid-like behaviour (Figs. 1 and 2, Table 3), which also correlates with the evolution of the scattering patterns and the apparent length (Fig. 3, Table 3). This could in part be attributed to the fact that EB24 is partly soluble in water (solubility of 0.5 wt.%). However, in the presence of 2 wt.% EB24, micellar growth still takes place at the same co-surfactant concentration (0.5 wt.% C12EO3) but a stronger rheological response is obtained. Therefore, the difference in behaviour between EC28, ECO26 and EB24 must be attributed mainly to the shorter EB24 fatty acid chain. The smaller oil requires less space inside the micelles, so that a higher concentration is required to produce an effect on the curvature strong enough to induce one-dimensional growth of the micelles. Longer fatty acid chains also induce the formation of longer worms, as indicated by longer relaxation times with EC28, compared to ECO26 and EB24 (Table 3) and longer apparent length (Table 3). Similarly, the comparison of ME17 and MC18 reveals a more efficient micellar growth with MC18, as seen from the longer relaxation times obtained (one-order of magnitude higher than with ME17) and longer apparent length (Tables 3). Considering now the effect of the alkyl chain length, the comparison of EC28 and MC18 reveals longer worms with MC18 (alkyl chain shorter by one carbon), as indicated by the higher zero-shear viscosity (550 Pa s compared to 11 Pa s in the case of EC28) and longer apparent length (720 Å compared to 670 Å in the case of EC28) . In the case of EB24 and BB44, which have a common fatty acid chain of four carbons, and a 2C difference in the alkyl chain, both were found to be less efficient in promoting wormlike micelles formation compared to the other (longer fatty acid) alkyl esters, however EB24 drives micellar growth at lower co-surfactant concentration (0.5 wt.% C12EO3) compared to BB44 (1 wt.% C12EO3). Overall our results show that the length of the fatty acid chain has a positive effect on promoting micellar growth, instead, the alkyl chain length has a negative effect. Finally, the effect of alkyl esters on the phase behaviour of ChEO10/C12EO3 micellar solutions can also be considered from the point of view of the position of the ester group for a fixed number

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of carbon atoms. ME17, ECO26 and BB44 all have 8 carbon atoms in total but differ in the position of the ester group along the chain: ME17 and ECO26 drive wormlike micelles formation in the absence of co-surfactant, compared to 1 wt.% C12EO3 in the case of BB44. In addition, the rheological parameters (G0, g0 and sR) and the micellar length up to 1 wt.% C12EO3 follow the order: ME17 > ECO26 > BB44. These results therefore confirm that shorter alkyl chain and longer fatty acid chain length promote wormlike micelles formation more efficiently. These results support the assumption that the alkyl (short) chain of the oil is probably located in the palisade layer of the micelles, along with the ester group; therefore shorter alkyl chains are more efficient in decreasing the average cross-section of the micelles and promoting their longitudinal growth. Instead, the fatty acid (longer) chain is more likely to be associated with the hydrophobic tails of the surfactant rather than forming an oil pool in the core of the micelles (which would have caused swelling of the micelles). Therefore the longer the chain, the more space required to accommodate it, decreasing the micellar curvature and driving wormlike micelles formation, but also leading to earlier phase separation in the presence of co-surfactant.

5. Conclusion The capacity of polyoxyethylene cholesteryl ether (ChEO10) micelles and their mixtures with triethylene glycol monododecyl ether (C12EO3) to encapsulate pharmaceutical oils was investigated, with the objective of correlating oil structure with macroscopic behaviour and final aggregate geometry, in view of further exploring the use of these systems as drug carriers. A range of alkyl ester oils with systematic changes in their structure were successfully solubilised: ethyl butyrate (EB24), ethyl caprylate (EC28), ethyl caproate (ECO26), methyl caprylate (MC18), methyl enanthate (ME17) and butyl butyrate (BB44). Unlike most hydrophobic alkanes which are usually reported to solubilise in the tails area and swell the micellar core [40], these oils were found to act as co-surfactants of smaller head-group, thus decreasing the surface curvature and promoting wormlike micelle formation. Rheological measurements showed a clear solid-like behaviour (G0 > G00 over a wide frequency spectrum and values as high as ca. 45 Pa) as well as a significant increase in the zero-shear viscosity (up to 700 Pa s) when adding a small amount of the alkyl esters at specific co-surfactant concentrations, thus confirming the presence of a viscoelastic network of wormlike micelles. SANS data confirmed a q1 behaviour typical of rods, and model fits to the scattering curves revealed rod-like structures with an unusual elliptical cross-section (with rminor 31 ± 2 Å and rmajor varying from 45 Å to 70 Å), as previously reported for these cholesterol-based surfactants but with another type of co-surfactant [15]. The formation of wormlike micelles in the presence of oils and the elliptical cross-section were also confirmed by Cryo-TEM images. The systematic variation in the molecular architecture of the oils (length of the chain on both sides of the ester linkage) and the combination of bulk and structural techniques enabled us to extract clear patterns in the packing of the oils and thus propose a rationale for their effect on aggregate shape, based on molecular structure. Alkyl esters with longer fatty acid chains for a fixed alkyl chain length were found to be more efficient in inducing the onset of wormlike micelles formation (i.e. at lower co-surfactant concentration) or their elongation (i.e. higher rheological parameters and longer micelles), following the order: EC28 > ECO26 > EB24 and MC18 > ME17. Instead, increasing the alkyl chain for a given fatty acid chain length had a negative effect on promoting micellar growth into worms (MC18 > EC28 and EB24 > BB44). Thus, comparing

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three oils with the same number of carbon atoms but a different position of the ester group showed that the onset of wormlike micelles formation followed the order: ME17 > ECO26 > BB44. SANS and Cryo-TEM measurements performed on turbid samples with co-surfactant concentrations beyond the zero-shear viscosity peak revealed the presence of more than one phase showing that, at least in the presence of oils, the loss of viscoelasticity and turbidity can be attributed to the transition to other structures than branched worms. In particular with EB24 oil, an unusual phase of tightly packed ring-like micelles was identified, which is attributed to the packing constraints imposed by the peculiar structure of the cholesterol-based surfactant. At high co-surfactant concentration, the addition of oil leads to either phase separation or the formation of lamellar aggregates at a lower co-surfactant concentration than in the absence of oil, probably due to the competition with the co-surfactant for the packing of the oil. One of the longer term motivations of this work is to examine the potential of wormlike micelles to solubilise drugs, and thus the impact of oil encapsulation on drug loading. Further experiments in our group have shown that solubilisation of these alkyl esters does not enhance the encapsulation of a model drug, namely, retinoic acid [47]. Based on the results presented here, this behaviour can easily be rationalised: these specific oils do not form an additional solubilisation pool in the micellar core and simply acts as co-surfactants. Interestingly, we found instead that the lengthening of the micelles into worms made a substantial improvement to drug solubilisation [47]; this result was also generalised to another wormlike micelle system based on an anionic surfactant (unpublished). Overall, our approach, which uses a systematic variation of the chemical architecture in a range of oils and a combination of bulk and microscopic techniques demonstrates the possibility of extracting general rules about the phase behaviour of wormlike micelles in the presence of oils. These rules bring precious insight into the packing of oils inside micellar aggregates as a function of their molecular structure and should form the basis of a formulation rationale when developing these systems for encapsulation applications. Acknowledgments H.A. and C.A.D. acknowledge ISIS and HMI (HZB) for the provision of beam time and the European Commission for financial support under the 6th Framework Programme through the Key Action: Strengthening the European Research Infrastructures (contract no.: RII3-CT-2003-505925 (NMI 3)). Beam line scientists Olivier Perroud (HMI), Sarah Rogers and Ann Terry (ISIS) are thanked for their help. H.A. acknowledges King’s College London for the award of a PhD studentship. The ARES rheometer used in this study was obtained on an EPSRC grant (EP/F037902/1). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcis.2012.04.014.

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