Janus emulsions stabilized by phospholipids

Colloids and Surfaces A: Physicochem. Eng. Aspects 441 (2014) 66–71

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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa

Janus emulsions stabilized by phospholipids Ildyko Kovach a , Joachim Koetz a,∗ , Stig E. Friberg b a b

Institut für Chemie, Universität Potsdam, Karl-Liebknecht-Strasse 24-25, D-14476 Potsdam, Germany Ugelstad Laboratory, NTNU, Trondheim, Norway

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• Janus

droplets containing olive oil/silicone oil/water. • Interfacial tension measurements using spinning drop and ring tensiometer. • Janus droplets stabilized by phospholipids. • Droplet size control by varying the viscosity of the oil components.

a r t i c l e

i n f o

Article history: Received 4 April 2013 Received in revised form 23 August 2013 Accepted 29 August 2013 Available online 7 September 2013 Keywords: Janus emulsions Spinning drop Interfacial tension Phospholipids

a b s t r a c t Janus emulsions were formed by mixing three immiscible liquids; this implies two oil components (i.e. olive oil (OO) and silicone oil (SiO)) with water in presence of interfacial active components. The morphology and size of Janus droplets formed strongly depended on the type of surfactant used. In presence of a non-ionic surfactant, i.e. Tween 80, large engulfed Janus droplets were formed. By adding phospholipids to the system the droplet size was decreased and more stable Janus droplets formed. Interfacial tension measurements carried out using a spinning drop apparatus and a ring tensiometer demonstrate that interfacial tension is the most important factor controlling the size, morphology and stability of Janus droplets. When the interfacial tension between oil and water becomes ≤1 mN/m, smaller Janus droplets are formed. Such conditions are fulfilled when phospholipids are used in combination with non-ionic surfactant Tween 80. The morphology of the double droplets is predominantly controlled by the viscosity and interfacial tension between the two oil phases. By using different types of phospholipids, i.e. asolectin and lecithin instead of a more concentrated phosphatidylcholine (phospholipon), the interfacial tension is decreased and different morphologies of engulfing can be observed. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Emulsions are colloidal systems with immense potential in different fields of application, i.e. in agriculture, medical, pharmaceutical, cosmetic, and food industry [1–3]. For stabilizing emulsion droplets the “classical” colloidal approaches can be used, i.e. electrostatic, steric, or electrosteric stabilization [4,5]. Furthermore, emulsions can be stabilized by adding colloidal particles, and so called “Pickering” emulsions are formed [6]. Another interesting

∗ Corresponding author. Tel.: +49 331 977 5220, fax: +49 331 977 5054. E-mail address: [email protected] (J. Koetz). 0927-7757/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfa.2013.08.065

stabilization mechanism is based on the formation of a liquid crystalline shell surrounding the droplets [7,8]. Nevertheless, the most important class of components used for stabilizing emulsions are surfactants due to the fact that the stabilization effect is accompanied by a decrease of the surface tension [1,2,9]. Therefore, the energy input can be minimized [1,2,9]. Besides the “classical” oil-in-water (o/w) emulsions “double” and “multiple” emulsions have reached great relevance in pharmaceutical and cosmetic industry, especially as drug delivery systems [10–12]. Recently, another type of emulsion was investigated in more detail, i.e. so called “Janus” emulsions with two non-mixable oil components dispersed in water into an o/w emulsion with both

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2.2. Preparation of Janus emulsions The surfactants and the dye were dispersed in oil (3 wt% PC, 2 wt% ASO at 40 ◦ C for 2 h in ultrasonic bath) and in water (4 wt% Tween 80, 2 wt% ASO, 2 wt% LEC) before the emulsification process starts. One gram emulsion contains 0.25 g olive oil, 0.25 g silicone oil and 0.5 g water. The emulsification was made in onestep in a 10 ml glass tube by mixing with Minishaker IKA (Roth®) at 2500 rpm. 2.3. Methods

Scheme 1. Different morphologies of Janus droplets.

oil components in one droplet [13,14]. The initial Janus emulsions were prepared by the extremely mild microfluidics method and the emulsions lent themselves to determining the effect of interfacial tensions on the drop topology as demonstrated by Guzowski [15]. Recently, Hasinovic and Friberg have shown that silicone oil (SiO) and vegetable oil (VO) form such Janus droplets in presence of a non-ionic surfactant, i.e. Tween 80, after high energy mixing the components with water [16–18]. Surprisingly the results indicated interfacial tension to play an important role, prompting a calculation of the correlation between drop topology and interfacial tension [19]. In general one can differ between different types of Janus droplets. Scheme 1 shows the different morphologies of engulfing from partially engulfed (B1, B2, B3) to completely engulfed (A1, A2): Nevertheless, in spite of strong indications of its importance, direct measurements of the interfacial tension in Janus emulsions are rare, and the stability of the Tween 80 stabilized Janus emulsions is limited. Therefore, the aim of the research was to improve the stability of the Janus droplets by adding another type of biocompatible surfactants, i.e. phospholipids. Spinning drop and ring tensiometer were used to measure the interfacial tension between the different liquid phases, i.e. water, olive oil (OO) and silicone oil (SiO). Microscopic investigations showed that phospholipids can significantly influence the droplet formation process in ternary oil–water–oil systems. Therefore, the biosurfactants lecithin (LEC), asolectin (ASO) and phospholipon 90G (PC), i.e. three conventional samples containing different amounts of phosphatidylcholine, were added to the oil–water–oil system. 2. Materials and methods 2.1. Materials 2.1.1. The following materials were used Silicone oil (SiO) of lower viscosity (10 mPa s) was obtained from Sigma-Aldrich® and silicone oil (SiO2) of higher viscosity (50 mPa s) from Polymerschmiede®. Olive oil (OO), and the olive oil soluble dye “Oil Blue N” were purchased from Sigma-Aldrich®. The water soluble Tween 80 (polyethyleneglycol sorbitan monooleate) and asolectin (ASO) (≈25% phosphatidylcholine from soybean) were supplied by Sigma-Aldrich®. Fat free lecithin (LEC) (≤10% phosphatidylcholine) was purchased from Carl Roth®. Phospholipon 90G (PC) ((≈94% phosphatidylcholine) was purchased from Phospholipon®. Methylrot was used as water soluble dye. Water was purified with a Milli-Q system (Millipore®).

2.3.1. Light microscopy Morphological and size analyses were done by using light microscopy (Leica® DMLB with a Lecia® DFC 295 live camera) and image analyser modul (Leica LAS Image Analysis). A drop of the emulsion was placed on a Roth® microscopic slide (76 × 26 mm) overlaid with a Roth® micro cover glass (20 × 20 mm). The microscopic images were captured at magnification 10 and 20. Afterwards the length of the Janus like drops was measured, and the data’s were transferred into a histogram. 2.4. Interfacial tension analyses 2.4.1. Ring method Interfacial tension () measurements between two immiscible liquids were examined with a Krüss® digital tensiometer K10TS using low vessel speed. The lower phase (30 ml) was taken into a glass pot. The platinum ring was cleaned in butane/propane flame and placed on the lower surface. Then the upper phase (15 ml) was placed on top of the lower phase. The sample was allowed to stay in the pot for 5 min and when the temperature was set to 25 ◦ C, the ring lifted up. The  values were read off directly from the apparatus in mN/m. 2.4.2. Spinning drop method In direct comparison to the above described ring method  was analyzed by means of a Dataphysics® SVT 20N Spinning Drop Tensiometer (SDT) (software SVT 20 IFT). To deliver , the density difference between the two phases was determined before. Density and refractive index data were obtained by using Anton Paar® DMA 4500 density meter combined with an Abbemat® RXA170 refractometer. The results are summarized in Table 1. The density difference also helps to find the best rotational speed, typically it is between 3000 and 6000 rpm. In practice the rotational speed must be chosen where the drop is ellipsoid and will not slip out of the frame. At first the capillary was bubble-free filled with the denser liquid (outer phase). Then the capillary (inner diameter 4 mm) was closed and inserted into the SDT. After this the inner phase with a lower density was injected with 1 ml syringe with a long needle (0.6 × 80 mm). The capillary was rotated (200 rpm) during the injection to prevent the contact between the drop (inner phase) and the capillary wall. With the proper rotational speed the drop was immobilized and the camera was calibrated by the camera movement. Afterwards a video was recorded and  was calculated [20]. 3. Results and discussion In Table 2 the different compositions of the investigated emulsions are summarized. Starting from the “classical” constitution of Janus emulsions (emulsion Nr. 1 and Nr. 2 in Table 2) according to Friberg and Hasinovic [16–18] (containing 4 wt% Tween 80) different amounts of phospholipids (PC, ASO, and LC) are added to the

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Table 1 Density and refractive index of the liquid phases at 25 ◦ C.

Density (kg/m3 ) Refractive index

Water

Water + 4 wt% Tween 80

Silicone oil (SiO)

Silicone oil SiO2

Olive oil

Olive oil + 3 wt% PC

Olive oil + 2 wt% ASO

0.99717 1.33265

1.00135 1.33849

0.93356 1.39922

0.95865 1.40215

0.90865 1.46689

0.91148 1.46979

0.91198 1.46751

Table 2 Composition of the emulsions. Emulsion number

Water

Olive oil

Silicone oil (SiO)

Silicone oil (SiO2)

1 2 3 4 5 6 7 8 9 10 11 * 12 13 * 14

X (+4 wt% Tween 80) X (+4 wt% Tween 80) X (+4 wt% Tween 80) X (+4 wt% Tween 80) X (+4 wt% Tween 80) X (+4 wt% Tween 80) X X X X X (+2 wt% ASO) X (+2 wt% ASO) X (+2 wt% LC) X (+2 wt% LC)

X X X (+3 wt% PC) X (+3 wt% PC) X (+2 wt% ASO) X (+2 wt% ASO) X (+3 wt% PC) X (+3 wt% PC) X (+2 wt% ASO) X (+2 wt% ASO) X X X X

X – X – X – X – X – X – X –

– X – X – X – X – X – X – X

* Janus emulsions are not formed. X Component incorporated. – Component not incorporated.

system. Taken into account that PC is only soluble in olive oil, LC only in water, and ASO in water and olive oil, different combinations in presence and absence of Tween 80 are made. One can see that in mixtures 3–10 PC or ASO, respectively, are solubilized in olive oil, whereas in mixtures 11–14 asolectin and lecithin are dispersed in water. Furthermore the viscosity of silicone oil is varied by using SiO in comparison to SiO2, respectively. It has to be noted here that the viscosity of olive oil is in the same order like silicone oil SiO2.

3.1. Light microscopy Our light microscopic investigations show that Janus-like emulsions are formed with the exception of emulsions Nr. 12 and 14. Taking into account that in presence of silicone oil of lower molar mass (emulsions 11 and 13) double oil droplets are formed, one can conclude that the viscosity of the two oil components also play an important role. Fig. 1 shows the micrographs of emulsions 1–6, this means emulsions with Tween 80 alone (emulsion 1 and 2) or in combination with PC (emulsion 3 and 4) or ASO (emulsion 5 and 6). Due to the presence of the olive soluble dye one can differ between the different oil components indicated exemplary on emulsion 2. Odd numbered emulsions contains low viscosity silicon oil, while those with even numbers silicon oil with higher viscosity. Emulsions formed in presence of Tween 80 by using SiO (emulsion 1) are completely engulfed and fulfill the requirements of a Janus emulsion very well. When SiO is substituted by SiO2 one can see that the droplet size is increased. Nevertheless, completely engulfed droplets of type A1 (compare Scheme 1) are still observed. Therefore, one can conclude that the droplet size strongly depends on the viscosity of the silicone oil. Additionally performed time dependent experiments show that the lifetime of the Tween 80 stabilized Janus droplets is limited. By means of a video performance one can see that the attachment of two Janus droplets can lead to coalescence when the two “olive” sides come in contact. It can be stated here to that the non-ionic surfactant Tween 80 at the concentration level used, is unable to stabilize Janus drops long term.

When asolectin is added to the olive oil (emulsion 5) significantly smaller, partially engulfed droplets are formed, which are detectable at higher magnification. When the silicone oil viscosity is increased (SiO2) the droplet size is increased, too, in good agreement with the results discussed above (Table 3). In presence of PC the effect is enhanced, that means the droplet size can be further decreased (Table 3), and the morphology of the individual droplets is more of the “B” type, this means of partially engulfed droplets. Nevertheless, a similar viscosity dependent size effect can be observed by substitution of SiO with SiO2. This implies the droplet size can be furthermore controlled by the viscosity of the silicone oil. In general one can conclude that the size decreasing effect of added phosphatidylcholin is accompanied by a stabilizing effect of the Janus droplets formed. It means that the vegetable oil top layer [18] is not observed in contrast to emulsion 1 and 2. For a more comprehensive discussion the influence of the different phospholipids in absence of Tween 80 was investigated. The results revealed the most far-reaching effect of viscosity on the emulsion topology; the drop size was radically smaller for the silicon oil of lower viscosity and a most strong variation in the drop size Table 3 Droplet size distribution of different emulsions. Emulsion number

Size range of the droplets (␮m)

Percentage of the measured droplets (%)

1

11–26 26–42 42–60 22–45 45–67 67–90 5–12 12–18 18–16 14–30 30–46 46–62 15–24 24–41 42–50

35 46 13 23 28 21 14 42 28 19 36 23 13 41 18

2

3

4

5

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Fig. 1. Micrographs of emulsions 1–6 (compare Table 2).

was observed (Fig. 2). Although more research is needed to clarify this effect, it is reasonable to emphasize the preferred solubility of PC in the olive oil makes it available both at the OO/W and OO/SiO interfaces. The contact angle is changed from the emulsions with Tween 80; it is now close to 90◦ .

3.2. Interfacial tension measurements First of all it has to be mentioned that interfacial tension measurements on Janus emulsions are not trivial. Therefore, we tried to measure the interfacial tension between the three liquid phases

separately by using two quite different methods, i.e. spinning drop and ring method. In Table 4 the results of ring tensiometry are summarized. The high values of interfacial tension between the oil–water phase was significantly decreased by adding Tween 80, this means from 14.3 to 2.9 mN/m for olive oil and from about 30 to 7.7 mN/m for silicone oil. By adding ASO or PC to the olive oil  is further decreased to 1.2 mN/m, and in combination with Tween 80 to 0.5 mN/m, demonstrating a synergistic effect between the non-ionic surfactant and phosphatidylcholine. By using asolectin the interfacial tension effect was quite independent from the way of incorporation into the aqueous or oil phase. The interfacial tension between

Fig. 2. Micrographs of emulsions 7–11, 13 (compare Table 2).

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Table 4 Interfacial tension given by the De Noüy tensiometer. Bottom phase

H2 O H2 O + 4 wt% Tween 80 H2 O + 2 wt% ASO H2 O + 2 wt% LEC *

Top phase OO (mN/m)

OO + 2 wt% ASO (mN/m)

OO + 3 wt% PC (mN/m)

SiO (mN/m)

SiO2 (mN/m)

14.3 ± 0.1 2.9 ± 0.1 1.2 ± 0.2 2.1 ± 0.1

1.2 ± 0.0 0.5 ± 0.1 – –

1.2 ± 0.1 0.8 ± 0.1 – –

32.9 ± 0.1 7.6 ± 0.2 8.7 ± 0.3 12.0 ± 0.2

29.8 ± 0.5 7.8 ± 0.3 * 2 ± 0.1 * 2.4 ± 0.2

Janus emulsions are not formed.

Table 5 Interfacial tension given by the spinning drop tensiometer. Outer phase

H2 O H2 O + 4 wt% Tween

Inner phase OO (mN/m)

OO + 2 wt% ASO (mN/m)

OO + 3 wt% PC (mN/m)

8 ± 0.4 3.2 ± 0.3

1.45 ± 0.05 0.45 ± 0.05

1.3 ± 0.5 1.05 ± 0.05

water and silicone oil is still higher, indicating a weaker effect. Only in presence of SiO2 a significant smaller value can be detected. However, in this case no Janus droplets are formed. The results obtained from spinning drop experiments are listed in Table 5. It can provide a good agreement with the results obtained by the ring method. The results bring forward two central factors that merit an analysis. At first the effect of viscosity of the silicon oil was unexpectedly large with the primary effect being larger drops for the oil with greater viscosity, but with the contact angles similar. This result accentuates the fact that the formation of emulsions [21] is a kinetic process and that drop division in the emulsification process [22] strongly depends on the viscosity of the dispersed phase. This correlation appears restricted to emulsions with combined surfactants. When only the water soluble Tween 80 is applied, Fig. 1 (emulsions 1 and 2), an approximately equal contact angle is found, but the low viscosity silicon oil forms substantially smaller drops, as mentioned. However, for the emulsions with mixed surfactants the contact angle varies strongly, ≈90 ± 60◦ , when different orientation is taken into account, Fig. 1 (emulsions 3–6). Similar variations are observed for emulsions stabilized by phospholipid surfactants, as to be seen in Fig. 2. Evidently, kinetic factors play a more significant role for systems with the latter surfactants, indicating the phospholipids as surfactants requiring longer times to reach a lower energy state than the single water soluble variety. Obviously, more research is needed to confirm this hypothesis, but in lieu of additional information, it is useful to point toward the fact that phospholipids form liquid crystals and, subsequent, vesicles during high energy treatment [23]. These processes in a water/oil system will also include transport over the interface with temporary interface structural changes, which latter may be affected by the interface per se and long term modification of the system is certainly expected [24]. More comprehensive investigation of the emulsification process is evidently needed. Additional support for the view that kinetics plays a more substantial role is the fact equilibrium calculations of the olive/silicon oil interfacial tension from the measured interfacial tensions, Tables 4 and 5, do not result in realistic values. Evidently, this discrepancy is not due to measurement inaccuracy; the agreement between the results from two different methods is excellent. Instead the possibility may be considered that slow transfer of surfactant between the three phases causes pronounced modifications of interfacial structure. Admittedly, such measurements would be time consuming in systems, in which all three liquids plus added surfactant are equilibrated, prior to measurement. A combination of such measurements with structural studies in more

concentrated systems may provide answers in an emerging difficult area of emulsion science. 4. Conclusions Our results show that the formation and stability of Janus emulsion droplets in the ternary olive oil/water/silicone oil system strongly depends on the interfacial tension at the oil/water interface. By using Tween 80, i.e. a biocompatible non-ionic surfactant, the interfacial tension between olive oil and water is decreased to 3 mN/m and completely engulfed Janus droplets are formed. When the viscosity of both oil components is of the same order (OO and SiO2) significantly larger droplets are formed. The droplet size can be decreased by lowering the viscosity of the silicone oil or by adding phospholipids. In presence of phosphatidylcholine the interfacial tension is decreased to 0.5 mN/m, and more stable Janus droplets are formed. Briefly examine the evidence one can assume that the phosphatidylcholine, embedded in the interfacial film between olive oil and water, stabilize the droplets against coalescence due to an additional electrostatic effect of the amphoteric head groups. The synergistic effect in presence of Tween 80 can be understood by the formation of a mixed film layer containing polyethylenglycol sorbitan monooleate and phosphatidylcholine. Based on similar ring tensiometer experiments Hameed et al. [25] concluded that Tween molecules are not tightly packed on the interface between olive oil and water, but might be in a tilted state. Therefore, one can assume in our case a more ordered structure in presence of phosphatidylcholine. Nevertheless, additional measurements are needed to characterize the orientation of the mixed surfactant film. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfa. 2013.08.065. References [1] S.E. Friberg, Some emulsion features, J. Dispersion Sci. Technol. 28 (2007) 1299–1308. [2] P. Becher (Ed.), Encyclopedia of Emulsion Technology. Volume 2: Applications, Marcel Dekker, Inc., New York, 1985. [3] N. Patel, U. Schmid, M.J. Lawrence, Phospholipid-based microemulsions suitable for use in food, J. Agric. Food Chem. 54 (2006) 7817–7824. [4] J. Sjöblom (Ed.), Surfactant Science Series. Volume 61: Emulsions and Emulsion Stability, Marcel Dekker, Inc., New York,·Basel,·Hong Kong, 1996. [5] R.J. Hunter, Zeta Potential in Colloid Science, Academic Press, London, 1981.

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