Preparation of Novel Silicone Multicompartment Particles by Multiple

21 Nov 2013 - Multicompartment poly(dimethylsiloxane) particles were produced for the first time using water-in-oil-in-water (W1/O/W2) emulsions as ...
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Preparation of Novel Silicone Multicompartment Particles by Multiple Emulsion Templating and Their Use As Encapsulating Systems Neus Vilanova,*,† Conxita Solans,† and Carlos Rodríguez-Abreu‡ †

Institute for Advanced Chemistry of Catalonia, Consejo Superior de Investigaciones Científicas (IQAC−CSIC) and CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Jordi Girona 18-26, 08034 Barcelona, Spain ‡ International Iberian Nanotechnology Laboratory (INL), Av. Mestre José Veiga, 4715-310 Braga, Portugal S Supporting Information *

ABSTRACT: Multicompartment poly(dimethylsiloxane) particles were produced for the first time using water-in-oil-inwater (W1/O/W2) emulsions as templates. Multiple silicone W1/O/W2 emulsions were successfully prepared by using silicone precursors with a low viscosity. Several formulation parameters were studied to determine their effect on the properties of emulsions and derived particles. It was observed that the mass fraction of the inner aqueous phase (φW1) and the concentration of both the hydrophobic and hydrophilic surfactants played a crucial role in the morphology and stability of the emulsions. Thus, the derived silicone porous particles also showed different characteristics depending on the emulsion formulation because of the templating effect. At low φW1 or high concentrations of the hydrophobic surfactant, particles showed smaller pore sizes as a result of more stable inner droplets. On the other hand, high concentrations of the hydrophobic surfactant resulted in an increase in the size of the derived particles, whereas high concentrations of the hydrophilic surfactant caused the opposite effect. In addition, fluorescein was encapsulated into the hydrophobic particles during the synthesis process and released in a controlled manner. The possibility to encapsulate simultaneously but independently two different hydrophilic components inside the same globule was also tested. On the basis of these results, the obtained silicone porous particles are envisioned to have applications in several advanced fields, for instance, as hydrophobic delivery systems.

1. INTRODUCTION Polysiloxanes, usually referred to as silicones, are inorganic polymers, made by [SiRR′O] repeating units, where R and R′ denote side groups, such as hydrocarbon chains or other reactive groups. Cross-linking reactions between reactive groups result in the formation of three-dimensional polymer networks, whose Si−O backbone and the chemical characteristics of the side groups endow the polymer with several interesting properties such as a low glass-transition temperature, low density, thermal stability, permeability to organic solvents and gases, high hydrophobicity or biocompatibility.1 Because of these particular properties, cross-linked silicones have been extensively used in various fields such as biomedical, water treatment, or material science, where they are processed in a wide range of formats, for instance as monoliths, films, membranes, or coatings.2−5 Nevertheless, the preparation of silicone-based particles with a controlled structure by means of soft colloidal templating has proved to be challenging.6−8 This arises from the fact that most common silicone precursors usually show rather high viscosities, typically between 500 and 4000 cSt, 9,10 which makes the emulsification process difficult.11,12 Hence, the preparation of silicone particles, particularly multicompartment, with a well-controlled structure © 2013 American Chemical Society

would open new interesting potential applications of silicones in many emerging fields such as controlled drug delivery, separation or enzyme immobilization. Generally, porous particles can be obtained using multiple water-in-oil-in-water emulsions as templates.13−15 Multiple water-in-oil-in-water emulsions (abbreviated as W1/O/W2) are compartmented systems that consist of dispersed oil globules which in turn contain smaller aqueous droplets. On account of their thermodynamic instability, multiple emulsions tend to phase separate through different destabilization mechanisms. Besides the general destabilization mechanisms of emulsions (creaming or sedimentation, flocculation, coalescence or Ostwald ripening),16 multiple emulsions also show other specific mechanisms, that mainly involve coalescence between the inner droplets, release of the the inner droplets towards the external continuous phase17−20 or diffusion phenomena; the latter essentially involves the migration of water molecules (arising from an osmotic pressure mismatch between the inner and the external phase) or the migration of the dissolved Received: August 14, 2013 Revised: October 28, 2013 Published: November 21, 2013 15414

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species driven by a concentration gradient.21 Multiple W1/O/ W2 emulsions are generally kinetically stabilized by the addition of hydrophobic and hydrophilic surfactants, which stabilize the inner aqueous droplets and the oil globules, respectively.22 As a result of the complexity of multiple emulsions, many formulation parameters are involved in determining their morphology and stability and consequently the characteristics of the emulsion-derived particles. Hence, formulation studies are required to investigate the broad range of types of particles that can be prepared. In this context, the present work addresses the formation of multiple W1/O/W2 emulsions using poly(dimethylsiloxane) precursors with a low viscosity to serve as templates for the preparation of porous particles. The structural changes in the emulsion induced by varying the concentration of an electrolyte in the inner aqueous phase, the mass fraction of the inner aqueous phase or the concentration of either the hydrophobic or hydrophilic surfactants were first systematically studied. Afterward, porous particles with different characteristics were obtained by cross-linking the intermediate oil phase through a thermally induced hydrosilylation reaction. It is worth to note that this cross-linking reaction is environmentally friendly and byproduct free. Additionally, this technique allowed the encapsulation within the pores of model hydrophilic substances that could be released in a controlled manner.

Table 1. Composition of the Standard Multiple W1/O/W2 Emulsion (EM_SF)a phases/ pseudophases primary W1/O emulsion

multiple W1/O/W2 emulsion

inner aqueous phase (W1) oil phase (O)

primary W1/O emulsion external aqueous phase (W2)

components water KF-6104 polymer mixture catalyst solution Those of primary W1/O emulsion 5 wt % Tween 80 aqueous solution

wt % 4 18 74 4 20 80

a

The polymer mixture was composed by PDMS and the cross-linker at a weight ratio of 30/70.

2.3. Determination of Cross-Linking Kinetics. The crosslinking reaction between the vinyl-terminated PDMS and the crosslinker was initiated by heating multiple emulsions up to 70 °C. The kinetic evolution of the cross-linking reaction in the emulsion was studied by gravimetry. The solid content at a certain time, namely the amount of cross-linked material, was determined by withdrawing an aliquot of 1 mL from the system. The multiple emulsion from the aliquot was destabilized adding 4 mL of a 1:1 (v:v) ethanol:acetone mixture. Afterward, the sample was centrifuged with the purpose of removing the supernatant. The obtained solid was washed twice with toluene to dissolve the un-cross-linked polymer and dried at 60 °C for 24 h. Finally, the remaining solid was weighed. The evolution of the cross-linking reaction was determined by calculating the degree of cross-linking for each aliquot, defined as

2. MATERIALS AND METHODS 2.1. Materials. The cross-linkable vinyldimethylsiloxy-terminated polydimethylsiloxane (abbreviated as PDMS) with a kinematic viscosity of υ = 0.7 cSt (MW = 186 g/mol) was supplied by ABCR (Germany). The cross-linker trimethylsilyl-terminated poly(dimethylsiloxane-co-methyl hydrosiloxane) (containing 50%mol of methylsiloxane) with υ = 12 cSt (MW=950 g/mol) was obtained from Sigma-Aldrich (USA). A Pt-based complex with the general formula platinum (0)-1,3-divinyl-1,1,3,3-tetramethyl-disiloxane from SigmaAldrich (USA), was used to catalyze the cross-linking reaction in a 9.7 wt % solution in chloroform. A polymeric silicone-based surfactant with side chains of glycerin groups, commercialized with the name KF6104 (HLB = 3−4), was a gift from Shin-Etsu (Japan). The sorbitan alkyl ethoxylated surfactant with the commercial name Tween 80 (HLB = 15) was supplied by Merck (Germany). These surfactants were used as hydrophobic and hydrophilic surfactants, respectively. NaCl used as a model electrolyte, Fluorescein and Pyronin Y as model hydrophilic substances and glucose as osmotic regulator for the encapsulation experiments, were purchased from Sigma-Aldrich (USA). All chemicals were used without further purification. 2.2. Preparation of Multiple Emulsions. Multiple W1/O/W2 emulsions were prepared following the two-step process. In the first step, the primary W1/O emulsion was preparing by dispersing the inner aqueous phase (W1) into the oil phase (O) in a tube of 1.5 cm of diameter using an Ultraturrax (stick S25N-10G, IKA) homogenizer at 13500 rpm for 3 min. The multiple W1/O/W2 emulsion was formed by dispersing the primary emulsion into a vial (2.7 cm of diameter) with the external aqueous phase (W2). In this second emulsification step, the system was gently stirred by a magnet at 1000 rpm in order to avoid the rupture of the oil globules. Preliminary formulation studies and a literature survey enabled us to select a reference emulsion composition for the multiple W1/O/W2 emulsion (see Table 1). This formulation will be referred hereafter as the standard formulation (EM_SF). Subsequently, on the basis of this EM_SF, several alternative formulations were prepared. All experiments were performed in duplicate. The formation and evolution of all multiple emulsions were followed by means of optical microscopy (Reichart Polyvar 2, LeicaMicrosystems). The size of globules and inner droplets was directly assessed from the optical microscopy images (averaged more than 1000 size measurements over each experiment).

degree of cross‐linking (%) = wt /wmc·100

(1)

where wt is the weight of solid at a time t and wmc is the weight of solid obtained when the network is considered to have reached a maximum cross-linking, which was assumed to occur after 24 h of reaction, as the obtained weight of solid remained practically constant. 2.4. Preparation of Porous Particles. Porous particles were prepared by cross-linking the intermediate phase of multiple W1/O/ W2 emulsions by heating them up to 70 °C. The dispersion was kept at this temperature and under stirring for 90 min (time determined by studying the cross-linking kinetics). Afterward, the obtained solid was washed 3 times with acetone using an ultrasound bath, and finally dried at 60 °C for 24 h. All experiments were performed in duplicate. The emulsion-derived particles were mainly characterized by scanning electron microscopy (SEM, Tabletop Microscope TM-1000, Hitachi). Particle and pore sizes distributions were directly assessed from SEM images (averaged over 1000 and 500 size measurements, respectively, over each experiment). 2.5. Preparation of Loaded Particles. Fluorescein loaded particles were prepared by dissolving the dye in the inner aqueous phase at a concentration of 0.007 M. To avoid water diffusion and thus maintain a stable multiple structure,21 the osmotic pressure of the external aqueous phase was matched to that of the inner droplets by adding glucose at a concentration of 0.014 M. The emulsion preparation, the cross-linking and washing processes were performed as described in previous sections. Particles encapsulating simultaneously Fluorescein and Pyronin Y in independent inner droplets were prepared following a similar process. For this particular experiment, two different primary W1/O emulsions were prepared separately, each one containing a solution of 0.007 M of the corresponding dye. Those primary emulsions were then emulsified at the same time with the external aqueous phase, which also contained glucose in order to match the osmotic pressure between both aqueous compartments. 2.6. Release Experiments. The encapsulated amount was determined by manually crushing 40 mg of dried loaded particles in a mortar. After dispersing the crushed particles in 3 mL of water for 1 week, the fluorescence of the supernatant was measured using a spectrofluorimeter Cary Eclipse (Varian). Samples were excited at a 15415

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wavelength of 470 nm, and the emission spectrum was recorded from 480 to 600 nm, showing a maximum at 513 nm. The amount of fluorescein was determined from a calibration curve. The encapsulation efficiency was calculated as the ratio between the experimental and the ideal encapsulated amounts, the latter being the initial amount dissolved in the emulsion template. Contact release experiments were performed by fixing 30 mg of fluorescein-loaded particles to a doublesided tape stuck to the bottom of a vial (note that each experimental point is one sample) and adding 3 mL of the receptor solvent (water or ethanol). Each vial was kept at 25 or 45 °C for different times without stirring. Finally, the fluorescence spectrum of the receptor solvent was measured. All experiments were performed in triplicate.

the presence of salts improves the stability of droplets as it helps to avoid coarsening phenomena.23 In these particular formulations, glucose was additionally dissolved in the external aqueous phase to prevent any osmotic mismatch between the internal and the external aqueous compartments. Multiple emulsions obtained adding 0.01 M of NaCl (Figure 1b) in the inner aqueous phase showed a similar morphology and stability than those obtained with EM_SF (Figure 1a). Conversely, using 0.1 M of NaCl resulted in unstable inner droplets, since they rapidly flocculated as shown in Figure 1c. As the addition of small amounts of salt did not show a significant improvement on the stability of the inner droplets, their use was ruled out for the following experiments. The influence of the inner aqueous phase was studied by preparing multiple emulsions with 5, 10, 18, and 30 wt % of mass fraction of the inner aqueous phase (φW1) with respect to the total polymer mixture weight. Note that the formulation with a φW1 of 5 wt % corresponds to the EM_SF. A change in φW1 essentially induced variations in the size and stability of the inner droplets. The mean size of the inner droplets increased from 2.8 ± 0.9 μm up to 4.4 ± 1.1 μm by increasing φW1 from 5 wt % up to 30 wt %. Besides, inner droplets of formulations with more than 5 wt % of φW1 appeared to be destabilized mostly by flocculation phenomena after 5 min of being prepared. Emulsions with 10 and 18 wt % φW1 showed quasispherical flocs (Figure 2a, b), whereas flocs from the system

3. RESULTS AND DISCUSSION 3.1. Formulation of Multiple Emulsions. The EM_SF resulted in a multiple emulsion stable for at least 90 min under stirring; its appearance under the microscope is shown in Figure 1a. Such emulsion showed an average globule size of

Figure 1. Optical micrographs of multiple emulsions with different concentrations of NaCl in the inner aqueous phase: (a) 0 M (EM_SF), (b) 0.01 M, and (c) 0.1 M after 5 min of being prepared. The diameter of the globules (Øglobule) at that time is also indicated.

47.9 ± 8.8 μm with inner droplets of 2.8 ± 0.9 μm. As multiple emulsions are rather complex systems, their stability and morphology are affected by numerous formulation parameters. Indeed, the properties of the primary emulsion, namely the size and stability of the inner droplets, are of utmost importance for the stability of the whole multiple emulsion. Hence, in the present work, several alternative formulations based on EM_SF were prepared, basically changing the formulation of the primary emulsion in order to study its effect on the stability of the multiple emulsion. The parameters studied were the electrolyte (salt) concentration in the inner aqueous phase, the mass fraction of the inner aqueous phase and the concentration of the hydrophobic surfactant KF-6104. Albeit the hydrophilic surfactant is ideally placed at the surface of the oil globules only, in reality both surfactants generally mix at both interfaces. Henceforth, the effect of the concentration of the hydrophilic surfactant Tween 80 on the characteristics of the multiple emulsion was also studied. Table 2 summarizes the parameters that were varied in the experiments. It is worth noting that one parameter was changed at a time, keeping the others constant. First, the electrolyte NaCl was dissolved in the inner aqueous phase at two different concentrations, 0.01 and 0.1 M. Usually,

Figure 2. Optical micrographs of multiple emulsions with different mass fractions of the inner aqueous phase (φW1): (a) 10, (b) 18 , and (c) 30 wt % after 5 min of being prepared. The diameter of the globules (Øglobule) at that time is also indicated.

with 30 wt % φW1 were nonspherical (Figure 2c). As regard of the oil globules, comparing Figures 2a−c, a slight increase of their size with φW1 is observed. It has been described that when φW1 is increased, the rheological properties of the primary emulsion change, basically its viscosity increases, which favors the formation of bigger globules.24 However, it is important to mention that after approximately 15 min, some of the inner flocs were released to the external aqueous phase, which favored the reduction of the globule size, eventually leading to globules with similar sizes regardless of φW1 (pictures not shown). Another strategy to modify the characteristics of the inner droplets is by changing the concentration of the hydrophobic surfactant KF-6104. Thus, formulations with 2, 5, 10, 20 and 25 wt % of hydrophobic surfactant (with respect to the total polymer mixture weight) were prepared. Note that the formulation with 20 wt % of hydrophobic surfactant corresponds to the EM_SF. The resulting emulsions with concentrations of 2, 10, and 25 wt % are shown in Figure 3a−c. In this particular case, pictures taken after 15 min are shown because at that time morphological differences between emulsions were more appreciable. Concerning the inner droplets, the lower the surfactant concentration, the bigger and the less stable the inner droplets were. Indeed, a rapid coalescence among the inner droplets was observed under

Table 2. Composition of EM_SF and Derived Formulations parameter varied

EM_SF

derived formulations

NaCl in W1 (M) inner aqueous phase (φW1) (wt %)a hydrophobic surfactant (wt %)a hydrophilic surfactant (wt %)b

0 5 20 5

0.01−0.1 10−30 2−25 2−20

a

Composition with respect to the weight of the polymer mixture. Composition with respect to the weight of the external aqueous phase (W2)

b

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Figure 3. Optical micrographs of multiple emulsions with (a) 2, (b) 10, and (c) 25 wt % hydrophobic surfactant KF-6104 after 15 min of being prepared. The diameter of the globules (Øglobule) at that time is also indicated.

Figure 5. Optical micrographs of multiple emulsions with (a) 2, (b) 10, and (c) 15 wt % hydrophilic surfactant Tween 80 after 5 min of being prepared.

36.2 μm (Figure 5a) down to less than 16 μm (Figure 5c) by increasing from 2 to 15 wt % the surfactant. In addition, the increase in the viscosity of the external aqueous phase by increasing the surfactant concentration would also give raise to the formation of smaller globules due to the high viscous shear applied. Nevertheless, it is also well-known that an excess of the hydrophilic surfactant also affects the stability of the inner droplets, as it diffuses from the external phase toward the interface of the inner droplets18,19,25 or it forms mixed micelles with the hydrophobic surfactant,17 eventually leading in both cases to less stable inner droplets. In the present system, by increasing the concentration of the hydrophilic surfactant above 5 wt % the inner droplets became unstable and they were progressively released, which also contributed to reduce the size of the oil globules. Figure 5a−c show that the number of inner droplets gradually decreased by increasing the concentration of the hydrophilic surfactant Tween 80. Summarizing this first part, it can be concluded that variations in the formulation of the primary W1/O emulsion as well as in the concentration of the hydrophilic surfactant, caused important changes in both the morphology and stability of the whole multiple emulsions, hence yielding templates with different morphological characteristics. 3.2. Preparation of Silicone Porous Particles. In general, mechanical properties of cross-linked materials depend on the structure of the network and the cross-linking degree.9,10,27 For that reason, it was important to study the cross-linking reaction that takes place in the intermediate phase. This study was carried out with the standard formulation EM_SF. Figure 6a displays the degree of cross-linking versus time at 70 °C. It can be deduced from the plot that the system achieved a high cross-linking degree after 90 min of reaction. Thus, to ensure particles with a highly cross-linked network, multiple emulsion templates were kept under stirring at 70 °C for 90 min. After cross-linking the intermediate oil phase of the EM_SF, silicone porous particles were successfully obtained. Scanning electron microscopy observations revealed that particles had a smooth surface (Figure 6b) and an inner structure that closely resembled that of the emulsion (Figure 6c), hence confirming the templating effect. Interestingly, the use of low viscous silicone precursors not only facilitated the emulsification process, but also provided stiffness to the resulting silicone network. The improved mechanical properties allowed obtaining poly(dimetylsiloxane) particles with a welldefined multicompartment structure upon drying, with no evidence of pore collapsing as observed in silicone-based capsules previously published.28 It should be pointed out that our attempts to obtain multicompartment particles using a commercial PDMS with higher viscosity (i.e., 4−8 cSt) were unsuccessful, which highlights the importance of selecting the right silicone precursor. Due to the templating effect, particles with diverse morphologies could be produced depending on the morphol-

optical microscopy for formulations with less than 10 wt % hydrophobic surfactant, resulting then in globules containing larger (about 5 μm) and fewer inner droplets (Figure 3a). With time, some of these big inner droplets reached the surface of the oil globule and were released to the external aqueous phase, which contributed to a reduction of the globule size. This agrees with the tendency of big inner droplets to be preferentially adsorbed onto the globule surface and released later on.25 Surprisingly, when an excess of the hydrophobic surfactant was used, a structure similar to an O/W/O/W emulsion was observed (Figure 3c). The excess of the surfactant might help to incorporate water from the external aqueous phase to the inner phase, producing such complex structure. Concerning the oil globules, an apparent dependence of its size with the surfactant concentration was detected, as it increased from 36.0 ± 6.1 μm up to 52.5 ± 9.3 μm (Figure 3a− c). The hydrophobic surfactant not only stabilized the inner droplets but also increased the viscosity of the primary emulsion, which as already mentioned, induced the formation of bigger multiple oil globules. Finally, the concentration of the hydrophilic surfactant Tween 80 in the external aqueous phase was changed from 2 up to 20 wt %. The primary W1/O emulsion used was that of the EM_SF. Multiple emulsions with 20 wt % Tween 80 could not be formed. This was attributed to the fact that the external aqueous phase was too viscous to properly emulsify the primary emulsion.26 On the other hand, formulations with a low concentration of Tween 80 seemingly needed longer emulsification times, since some aqueous droplets were not incorporated inside globules instantaneously as pointed by the arrows in Figure 4. Comparing Figure 5a−c, it can be concluded that the concentration of the hydrophilic surfactant Tween 80 had a great effect on the globule size as it decreased from 126.2 ±

Figure 4. Optical micrograph of a freshly prepared multiple emulsion with 2 wt % Tween 20. The arrows point to the primary W1/O emulsion not well dispersed in globules. 15417

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Figure 6. (a) Evolution of the degree of cross-linking with the reaction time for EM_SF. (b, c) Scanning electron microscopy pictures of whole (b) and sectioned (c) particles obtained by cross-linking EM_SF.

ogy of the emulsion template used. Although both particle and inner pores size show a broad size distribution, it was possible to establish trends that give information about the influence of emulsion formulation parameters on the properties of particles. The effect of emulsion φW1 on silicone particle size and morphology is shown in Figure 7. Emulsion templates with a low φW1 resulted in particles with a smooth surface, while templates with a φW1 above 10 wt % resulted in particles with a porous surface (Figure 7a). These pores were mainly attributed to the release of inner flocs during the cross-linking reaction. Comparing Figure 6c (φW1 = 5 wt %) with Figure 7b, c (φW1 = 18−30 wt %), it is also visible a progressive loss of the spherical shape of the inner pores by increasing φW1. This change was also accompanied with a significant increase of pore polydispersity, together with a pore size increase from 2.5 ± 1.1 μm up to more than 7 μm (pore size could not accurately measured in this case because of the nonspherical shape of the pores) (Figure 7d). All these trends were consistent with the existence of bigger and less stable inner droplets upon increasing φW1. In general, particle size of all samples was between 40 and 45 μm. Concerning the effect of the concentration of the hydrophobic surfactant KF-6104, although spherical particles with a smooth surface were always obtained in the explored concentration range, important changes in both, particle and pore size were observed. As the stability of the inner droplets in the templating emulsions increased with the concentration of hydrophobic surfactant, the resulting particles showed a gradual increase in the number density of the pores (Figures 6c and 8a, b), together with a decrease in its polydispersity and size, the latter down to a minimum around 2.5 μm (Figure 8d). However, a drastic increase in pore size was detected when using an excess of above 20% of the hydrophobic surfactant (Figure 8c, d), mainly because of the formation of a much more complex multiple emulsion. Regarding the particle size, it

Figure 7. (a−c) Scanning electron microscopy pictures of (a) whole and (b, c) sectioned particles obtained from multiple emulsion templates with 18 wt % and 30 wt % of φW1. (d) Influence of φW1 on particle and pore size. The pore size of particles with φW1 = 30 wt % is just an approximate value. For comparison, the size of the emulsion globules 5 min after preparation is also shown. Dashed lines are only a guide.

increased from 28.8 ± 7.4 μm up to 44.4 ± 9.3 μm by increasing the concentration of the hydrophobic surfactant KF6104 (Figure 8d), in agreement with the increase in the emulsion globule size due to viscosity effects. Concerning the particles obtained from templates with different concentrations of the hydrophilic surfactant Tween 80, all of them presented a spherical shape and smooth surface. Because the system with 15 wt % Tween 80 presented only few multiple globules (Figure 5c), that formulation was not considered for the synthesis of particles (gray zone in the plot in Figure 9c). Due to the instability caused by the excess of the hydrophilic surfactant, the number density of pores gradually decreased by increasing the concentration of hydrophilic surfactant (Figure 9a, b), which is attributed to the release of the inner droplets in the corresponding emulsion templates. The pore size, however, remained almost constant in all particles. As expected, the concentration of the hydrophilic surfactant was critical for regulating the particle size, which drastically decreased from around 100 μm down to 13 μm, by increasing the surfactant concentration from 2 to 10 wt % (Figure 9c). The experiments reported here thus confirm that the characteristics of the particles depend on both the stability and morphology of the initial multiple emulsion templates. 15418

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Figure 9. (a, b) Scanning electron microscopy pictures of sectioned particles obtained from multiple emulsion templates with 2 and 10 wt % hydrophilic surfactant Tween 80. (c) Influence of the concentration of the hydrophilic surfactant on particle and pore size. For comparison, the size of the emulsion globules 5 min after preparation is also shown. Dashed lines are only a guide. Figure 8. (a−c) Scanning electron microscopy pictures of sectioned particles obtained from multiple emulsion templates with 2 , 10, and 25 wt % hydrophobic surfactant KF-6104. (d) Influence of the concentration of the hydrophobic surfactant on particle and pore size. For comparison, the size of the emulsion globules 15 min after preparation is also shown. Dashed lines are only a guide.

Figure 10. Schemes of different scenarios observed in multiple emulsion templates (left), which lead to particles with different characteristics (right).

Figure 10 summarizes the different templating effects observed that can be tuned by emulsion formulation parameters. In general, in all systems, particles were on average about 20% smaller than the original globules, as a result of the cross-linking and drying processes. Such reduction can be noticed by comparing the plotted globule sizes with the corresponding particle sizes in Figures 7−9. 3.3. Porous Particles As Encapsulating and Release Systems. The synthesis of silicone porous particles via multiple emulsion templating allows the encapsulation of substances with a low compatibility with the polymer matrix (i.e., hydrophilic molecules) during the synthesis process, with no need of a second encapsulation step, as suggested by earlier works.28 This approach is difficult to reach via simple emulsion templating. To prove the one-step encapsulating ability using the multiple emulsion strategy, Fluorescein was chosen as a model hydrophilic molecule. The formation of the loaded multiple emulsions and their cross-linking process were followed by fluorescent optical microscopy. The appearance of fluorescence in the external aqueous phase evidenced a slight diffusion of the encapsulated Fluorescein toward the external phase, essentially due to a concentration gradient.21 The loading efficiency of the system was around 18%. Although it is in line with similar previous publications,29 such a low loading

efficiency was mainly attributed to the washing process, as the washing liquid (acetone) acts as a swelling agent and facilitates the leakage of the dye, as commented below. Therefore, the loading efficiency could be improved by optimizing the washing process. Figure 11a clearly shows that Fluorescein is encapsulated inside the pores of the particles. The similarity between dye-loaded and nonloaded particles evidenced that the effect of Fluorescein on particle morphology was minimal and that the osmotic pressures were properly matched (no water diffusion). Release experiments using water as a receptor solvent at two temperatures are shown in Figure 11b. The release pattern at both temperatures was similar: an initial burst followed by a decrease in the release rate. However, it is evident that fluorescein was released significantly faster at 45 °C than at 25 °C. Indeed, at 45 °C almost a total release was achieved after 144 h (6 days), whereas at 25 °C, less than 50% of the fluorescein was released within the same time. The dissimilarity between the release rates could be attributed to an increase in the diffusion coefficient and solubility of the fluorescein in water with temperature. The same release experiments were carried out using ethanol as a receptor solvent. In this particular case, the release was much faster than in water, as after 4 h all fluorescein was 15419

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Figure 11. (a) Fluorescence optical micrograph of fluorescein-loaded particles. (b) Release profiles at 25 and 45 °C of the encapsulated fluorescein using water as a receptor solvent. Solid lines are a fitting to eq 2.

Figure 12. (a) Release profiles at 25 °C of the encapsulated Fluorescein using ethanol as a receptor solution. Solid line is a fitting to eq 2. (b) Optical micrographs of a particle dried and swollen with ethanol.

process. Moreover, in the particular case of using ethanol, the solvent modifies the morphology of the particles along the release process, which is not contemplated in the applied model. These results demonstrate that the permeability of the solvent through the matrix as well as the structure of the polymer network play an important role in the release process. One additional advantage of the multiple emulsion method is that, in principle, it is possible to encapsulate simultaneously different components inside the same globule but in separate inner droplets. For that, the diffusion of such components between inner droplets has to be minimized. As a proof-ofconcept, we encapsulated simultaneously Fluorescein and Pyronin Y, by preparing separately two W1/O emulsions containing each of the dye inside the aqueous droplets. Those emulsions were then emulsified to obtain W1/O/W2 emulsion with both dyes inside the globules. The very low solubility of the dyes in the intermediate oil phase appeared to slow down interdroplet diffusion so that inner droplets with distinct fluorescence emission were detected just after formation of globules (see Figure S1 in the Supporting Information); dye mixing was however observed after ca. 20 min. The ability of such double encapsulation in multiple emulsions has already been proved using microfluidic devices.35,36 However, this preliminary test shows the possibility of simultaneous and independent encapsulation of different components using a

released (Figure 12a). This fast release arises from the swelling action of the ethanol over the silicone network,30 as it is evidenced in Figure 12b. The swollen state of the silicone network might facilitate the diffusion of the fluorescein through the matrix. To describe the experimental data, we considered a release model for spherical structures based on Fick’s law31 ⎡ 6 % released = ⎢1 − 2 ⎢⎣ π

∞

∑ n=1

⎛ Dn2π 2t ⎞⎤ 1 exp ⎜− ⎟⎥ × 100 n2 ⎝ R2 ⎠⎦⎥ (2)

where n is the number of terms in the series (n = 6), D is the diffusion coefficient (fitting parameter) of the Fluorescein in the silicone matrix, and R the radius of the particle. This equation has already been used to describe release patterns for various systems such as core−shell structures or polymer particles.32,33 From the fittings to eq 2, the diffusion coefficients for the release in water at 25 and 45 °C and in ethanol resulted in 3.36 × 10−13, 4.78 × 10−13, and 1.92 × 10−11 m2/h, respectively, which are within the same order of magnitude of published data for fluorescein encapsulated in other matrices.34 However, these values are only indicative, as the experimental data could not be properly fitted to the release model (solid lines in Figures 11b and 12a). This might indicate that other mechanisms besides diffusion could contribute to the release 15420

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much more simple bulk emulsification technique (which does not require any special device), although much additional work is required regarding proper formulation and cross-linking of such systems.

4. CONCLUSIONS Silicone multiple W1/O/W2 emulsions with different characteristics were formed for the first time using silicone precursors with a low viscosity. Giving that there was no need to further modify the rheological properties of the intermediate oil phase, for instance by adding an organic solvent, the intermediate oil phase was almost totally converted into the silicone network. Consequently, the obtained porous silicone particles mimicked the characteristics of the corresponding emulsion templates. It was found that the addition of a salt in the inner aqueous phase did not have a great influence on the behavior of the emulsion, unless it is used in excess, which causes destabilization. On the other hand, the mass fraction of the inner aqueous phase and the concentration of the hydrophobic surfactant had a large influence on determining the final size, morphology and number density of the inner pores. Thus, it can be concluded that the characteristics of the primary emulsion effectively determines most of the features of the derived multiple emulsions. Conversely, the hydrophilic surfactant mainly affects the particle size and the number density of the pores. Porous silicone particles showed to be interesting candidates for temperature and solvent activated molecular delivery applications. Systematic studies on the properties of the present silicone particles will be further reported elsewhere.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

* Supporting Information S

(S1) Optical and fluorescent micrographs of multicomponent multiple emulsions. This material is available free of charge via the Internet at http://pubs.acs.org.

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the Ministerio de Economia y Competitividad (Grant CTQ2011-29336-C0301/PPQ) and Generalitat de Catalunya (Grant 2009 SGR-961) ́ is grateful to the for the financial support. C. Rodriguez-Abreu European Union’s Seventh Framework Programme (FP7/ 2007-2013) under COOPERATION program NMP-theme (Grant 314212) and Xunta de Galicia (PGIDIT, 2010/PX168) for research funding. N. Vilanova thanks CSIC for a JAEpredoctoral scholarship.

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