W/O/W EMULSIONS OBTAINED FROM SELF EMULSIFIYNG BASES AND FATTY ALCOHOLS SHOWING LAMELLAR LIQUID CRYSTALS ROCHA-FILHO, PA; VIEIRA, GS; BORTOLIN, ME; MORAES, CSS Department of Pharmaceutical Sciences. Faculty of Pharmaceutical Sciences of Ribeirão Preto. University of São Paulo. Ribeirão Preto, SP. Brazil pedranjo@fcfrp.usp.br

Emulsions are dispersed multiple phase systems. They are made out of at least two nearly immiscible fluids, one being dispersed in the other. The dispersed phase forms droplets, which are surrounded by the continuous phase [1]. Multiple emulsions are very complex systems as the drops of dispersed phase themselves contain even smaller droplets, which normally consist of a liquid miscible and in most cases identical with the continuous phase [2, 3]. Multiple emulsions are defined as emulsions in which both types of emulsions, i.e. water-in-oil (W/O) and oil-in-water (O/W) exist simultaneously [4]. Both hydrophilic and lipophilic emulsifiers are used for the formation of multiple emulsions. They combine the properties of both W/O and O/W emulsions. These have been described as heterogeneous systems of one immiscible liquid dispersed in another in the form of droplets, which usually have diameters greater than 1µm [4]. These two liquids forming a system are characterized by their low thermodynamic stability [5]. Owing to the liquid membrane structures multiples emulsions have many potential applications in such diverse fields as pharmaceutics, cosmetics, food, and separation technologies [5]. In cosmetics, multiple emulsions are useful when one wishes to prepare sustained release aerosol fragrances, prolonged skin moisturizers and protection of sensitive biologicals, personal care formulations for perfumes, skin lipids, vitamins, and free radical scavengers [6]. However, compared with simple emulsions consisting of only two phases, much more destabilization processes need to be taken into consideration for multiple emulsions [7].

In the development of multiple emulsions, the following variables are important and should be considered: (i) primary emulsifier, (ii) nature of the oil phase- various paraffinic oils are preferred, (iii) secondary emulsifier (iv) secondary volume fraction, that is, between 0.4 and 0.8 depending on required viscosity, (v) thickeners or additives, and (vi) processing of primary emulsion at high shear mixing while of secondary emulsion at low shear mixing [7].

The aim of this study was to compare characteristics of W/O and W/O/W emulsions formulated with similar ingredients. The physical properties of these two types of vehicles were characterized first. The multiple emulsions were prepared by the two-steps method. MATERIALS AND METHODS

a) To develop W/O emulsion Oil phase: different blends of solid paraffin and liquid paraffin (Table 1). Primary surfactant: sorbitan monooleate (Span® 80) (HLB=4.3) polyoxyethylene (20) sorbitan monooleate (Tween® 80) HLB= 15.0) (both by Oxiteno) at different HLB values in the range of 5.0 to 8.0. Different primary volume fraction (ΦW/O) from 0.25 to 0.40, were essayed to obtain most stable W/O primary emulsion.

Table 1. Oil phase composition (w/w %).

Solid paraffin Liquid paraffin

10 40

20 20

30 10

40 10

b) To develop the multiple emulsions A) Choice of secondary surfactant: 1) Ethoxylated Fatty Esters: PEG- 20 Sorbitan Monolaurate (Tween® 20) (HLB=16.7); PEG-20 Sorbitan Monostearate (Tween® 60) (HLB=14.9); PEG-20 Sorbitan monooleate (Tween® 80) (HLB=15.0); Trilaureth-4 Phosphate (Hostaphat® 340); 2) Self Emulsifying Base (SEB): Cetearyl Alcohol and Ceteareth-20 (Cosmowax® J) (Croda do Brasil); Fatty alcohol, Mono-, Di-, and Tri-(alkyltetraglycolether)-o-phosphoric acid esters, sodium cetearyl sulfate and fatty alcohol polyglycol ether (Hostacerin® CG) (Clariant). B) Preparation of W/O/W Type multiple emulsions: Primary emulsion was prepared by emulsifying the oil phase containing hydrophilic and lipofilic surfactant oil with aqueous phases heating both phases at 75± 1oC. Aqueous phase was slowly added to the oil phase while stirring at 500 rpm. Agitation was continued until cooling to room temperature of 25± 2oC. Resultant simple emulsions with varying compositions of oil and emulsifier were stored at room temperature and each sample was subjected to centrifugation and microscopy evaluation to observe liquid crystal formation. All the multiple emulsions have been produced by a two-steps emulsification strategy. For multiple emulsions development several pre-formulation studies were performed initially using different concentrations of W/O emulsions. The primary emulsion obtained was subjected to second stage emulsification : then, for obtaining the multiple emulsion, primary emulsion was slowly added in different primary volume fraction to the aqueous phase containing hydrophilic secondary surfactant while agitating at 500 rpm for 30 min. At this time multiple emulsions were confirmed by microscopy. Time of W/O addition, homogenization time and concentration of hydrophilic emulsifiers were varied to obtain a multiple emulsions. Briefly, simple emulsion was produced by emulsifying the oil with the lipophilic emulsifier and the mixture was preheated at 75±5°C before the emulsification. Mixing of the aqueous phase with the oil phase continued at 500 rpm for 30±2min. For the second stage emulsification, the simple emulsion was added to the aqueous phase containing hydrophilic emulsifier heated at 75±5°C. A stirring speed of 350 rpm was maintained until the formation of multiple emulsions which was confirmed by microscopic analysis. Characterization of multiple emulsions Properties of primary and multiple emulsions Types of emulsions Types of emulsions were analyzed by dilution with paraffin oil and water separately and observation under microscope.

Microscopic tests Multiple emulsions were analyzed under polarizing microscope (Olympus BX 50). to confirm the multiple characters. A drop emulsion was placed on the glass slide without dilution and covered by a glass cover. A drop of immersion oil was placed on the cover slide and observed under the microscope. Centrifugation tests Centrifugation tests (centrifuge Excelsa Baby II - Fanem Ltd., São Paulo, Brazil) were performed for the primary and multiple emulsions 24 hours after preparation. The same test was repeated for the multiple emulsions after 24 h, 3 days, 7 days, 14 days and 28 days of preparation. Centrifugation conditions: 5 g of sample were put in the tube at a 25± 2oC and 3000 rpm for 30 minutes. pH determination The pH values of the freshly prepared multiple emulsions and the samples emulsions kept at different conditions were determined by a pH meter Analion-(Mod. PM608). pH measurements were repeated for multiple emulsions after 1, 3, 7, 14, 21 and 28 days of preparation. Electrical conductivity tests Conductivity tests were performed for the multiple emulsion immediately after preparation and for the samples kept at different conditions by using conductivity-meter. Tecnopon (mCA Model 150). Conductivity tests were repeated for the multiple emulsions after 1, 3, 7, 14, 21 and 28 days of preparation. Stability studies For the stability studies, simple and multiple emulsions were weighed (50g) and packed into glass containers with 100g capacity and were kept at different storage temperature for a period of 30 days at different storage conditions: (25±2°C), (4±2°C) and (40±2°C). RESULTS AND DISCUSSION The blend of solid paraffin and liquid paraffin oil (1:4) with fatty esters at HLB value of 5.0 and Ø W/O = 0.25 allow obtention of stable W/O emulsions (Table 2). This W/O primary emulsion showing lamellar liquid crystalline phase (Figure 1) was employed to obtain W/O/W emulsion. Table 2. W/O composition.

(%) (w/w)→ Components↓

Oil phase: Solid paraffin Liquid paraffin

50.0 10.0 40.0

60.0 12.0 48.0

70.0: 14.0 56.0

80.0: 16.0 64.0

Water phase 45.0 35.0 25.0 15.0

Surfactants: Span® 80

Tween® 80

5.0: 4.68 0.32

5.0: 4.68 0.32

5.0: 4.68 0.32

5.0: 4.68 0.32

Φ W/O 0.45 0.35 0.25 0.15

24hours

1 month

Figure 1. Photomicrography W/O showing lamellar phase. [Primary emulsion (ΦW/O= 0.25); HLB=5.0; (photomicrography polarized -200X)]

W/O emulsion is a cosmetic base that makes oily constituents efficiently spread on the skin surface to provide high emollience and others functions. The conventional W/O emulsifiers which stabilize the W/O emulsion at the oil- water interface, tend to be dissolved as a monodispersed molecule in oil bulk due to the lack of lipophobicity causing inefficiently stabilization (8). W/O emulsions maintained in different stockage conditions show phase separation after one month, if we consider the presence of liquid crystals. To develop the multiple emulsions A) Choice of secondary surfactant: 1) Ethoxylated Fatty Esters:

Composition %w/w

Primary emulsion 20- 45

Aqueous phase 50- 75

Secondary surfactant: Ethoxylated Fatty Esters or

Self Emulsifying Base (SEB)or [Ethoxylated Fatty Esters+

Self Emulsifying Base (SEB)]

0.5-5.0

Similarly to Kavaliunas and Frank (9) we use photo micrography to detect the structure of simple and complex emulsions. T. Ito et al (10) prepared W/O/W- type multiple emulsions containing lamellar liquid crystal by mixing a W/O-type emulsion (prepared by primary emulsification) with a lamellar liquid crystal obtained from poly(oxyethylene) stearyl ether, cetyl alcohol, and water, and by dispersing and emulsifying the

mixture in an outer aqueous phase. Fatty alcohols were used in both steps in our research: for W/O and W/O/W emulsion. Only for W/O emulsion were observed lamellar phase with fatty esters. Analysis microscopic don´t show anisotropy for W/O/W. Others blends of oil primary phases and W/O volumetric fraction should be also studied. For the proposed oil blend (solid paraffin: liquid paraffin -1:4) some observations can be described. Multiples globules of different size were obtained with PEG fatty alcohols (Figure 2) sometimes empty or little inner content. SEB produces multiples globules almost full and reduced size (Figure 3). The association of PEG- fatty alcohols (especially Tween® 20 and Tween® 80), with self emulsifying bases allow multiple emulsion showing full globules (Figure 4). The secondary surfactants concentrations association (PEG- fatty alcohols+ SEB) for W/ O/W system needs further study, as well as the conditions (temperature, agitation time, agitation speed and time of W/O addition represent the priority of our future studies. Figure 5 shows anisotropic structures for external aqueous phase with different surfactants and we can see lamellar phase for Tween® 20 for Cosmowax® J and Tween® 60 for Hostacerin® CG..

CONCLUSION

Multiple phase emulsions are increasingly used as alternatives to simple emulsions in personal care products. One of the major advantages of these emulsions over simple emulsions is slow and controlled release of their ingredients. Fatty alcohols employed as secondary surfactants promote formation of W/O/W emulsion with a short life time. Association of fatty alcohols wit self emulsifying bases can increase the life time of multiple globules. Our next step is to study the quantities secondary surfactant and the better process condition to obtain stable W/O/W emulsions.

acknowledgments Alberto Gomes (Universidade Federal do Amapá- BR) ; Lara Meloni e Patricia Tagava ( Universidade de São Paulo- FCFRP); Thiago Mubárack (Universidade Federal do Maranhão-BR)

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Tween® 20 Tween® 60 Tween® 80

200X

400X (immersion)

Figure 2-. W/O/W fatty esters as secondary surfactant.

Hostaphat® kd 340 Hostacerin® CG Cosmowax® J

200X

400X (immersion)

Figure 3. W/O/W SEB as secondary surfactant.

Figure 4. W/O/W SEB associated to PEG fatty esters as secondary surfactant (200X). 1) Cosmowax® J; 2) Cosmowax® J+ Tween® 20; 3) Cosmowax® J+ Tween® 60; 4) Cosmowax® J+ Tween® 80.

Cosmowax® J+ Tween® 80 (200X)

Cosmowax® J+ Tween® 20 (200X)

Cosmowax® J+ Tween® 60 (200X)

Hostacerin® CG+Tween® 60 (400X)

Figure 5. Microscopic analysis of external aqueous phase.