Preparation of non-aqueous Pickering emulsions using anisotropic block copolymer nanoparticles
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In this work, we show that amphiphilic diblock copolymer worms prepared via alcoholic RAFT dispersion polymerization can be used to stabilize non-aqueous Pickering emulsions. A previously reported synthesis protocol based on polymerization-induced self-assembly (PISA) was modified to enable the preparation of poly(2-(dimethylamino)ethyl methacrylate)-poly(benzyl methacrylate) (PDMA-PBzMA) worm-like particles directly in methanol at relatively high solids. A dilute dispersion of these highly anisotropic nanoparticles was then homogenized with sunflower oil to produce sunflower oil-in-methanol emulsions. The mean droplet diameter ranged from 9 to 104 μm, depending on the nanoparticle concentration and the stirring rate used for homogenization. The sunflower oil content was increased systematically, with stable emulsions being obtained up to a volume fraction of 0.60. In all cases, the sunflower oil droplets gradually increase in size on ageing for up to 4 days. However, stable emulsions were obtained after this time period, with no further change in the mean droplet diameter for at least 2 months on standing at ambient temperature. Turbidimetry studies of the continuous phase after sedimentation of the relatively dense emulsion droplets indicated that the initial adsorption efficiency of the PDMA-PBzMA worms is very high, but this is reduced significantly as the droplet diameter gradually increases during ageing. There is a concomitant increase in fractional surface coverage over the same time period, suggesting that the increase in droplet diameter is the result of limited coalescence, rather than an Ostwald ripening mechanism.
KeywordsRAFT polymerization Self-assembly Pickering emulsions Non-aqueous emulsions Block copolymers Nanoparticles
A Pickering emulsion comprises either oil or water droplets that are stabilized by solid particles adsorbed at the oil/water interface. This type of emulsion was first reported by Ramsden over a century ago  but Pickering’s subsequent studies received more credit and led to today’s nomenclature . Various classes of solid particles have been employed as Pickering emulsifiers, including silica [3, 4, 5, 6, 7], inorganic clays [8, 9, 10, 11, 12], and organic polymer latexes [13, 14, 15, 16, 17, 18]. Either oil-in-water (o/w) or water-in-oil (w/o) Pickering emulsions can be obtained, with the emulsion type mainly depending on the contact angle (θ) between the particles and the interface. Thus, hydrophilic particles possess contact angles less than 90° and reside in the aqueous phase, favoring the stabilization of o/w emulsions. In contrast, hydrophobic particles are characterized by contact angles greater than 90° and hence are preferentially located within the oil phase, resulting in the formation of w/o emulsions.
The main driving force for the interfacial adsorption of particles is the reduction in surface area. The energy required to detach spherical particles adsorbed at the interface is dictated by the contact angle and the particle radius [19, 20]. In many cases, larger particles can be considered to be essentially irreversibly adsorbed, since the detachment energy is several orders of magnitude greater than the particle thermal energy [19, 20]. Since the adsorbed particle layer prevents droplet coalescence by providing a strong steric barrier, Pickering emulsions tend to be far more stable than surfactant-stabilized emulsions . Moreover, Pickering emulsions also offer several other advantages, such as reduced foaming during homogenization, more reproducible formulations, and lower toxicity [20, 21].
Although far less commonly reported than w/o or o/w emulsions, there are various examples of non-aqueous emulsions in the literature [22, 23, 24, 25, 26]. Such systems require a pair of immiscible solvents . In principle, non-aqueous emulsions could be utilized for water-sensitive reactions or polymerizations [26, 28, 29], for reactions that need to be conducted above the normal boiling point of water  or for specific applications where the presence of water is problematic [31, 32].
In 2004, two research groups led by Paunov and Velev reported the formation of colloidosomes, Pickering emulsions, and foams stabilized using “polymeric microrods” [33, 34], rather than conventional spherical particles. These microrods were prepared from epoxy-type photoresist SU-8 using the liquid-liquid dispersion technique and possessed relatively large dimensions (mean rod length = 23.5 μm; mean rod width = 0.6 μm). More recently, we have described the use of much smaller hydrophilic diblock copolymer worms as Pickering emulsifiers for the preparation of o/w  or w/o  emulsions. In principle, such highly anisotropic particles should be more strongly adsorbed than precursor spherical particles (i.e., whose mean diameter is comparable to the mean worm width). Other research teams have also reported that anisotropic particles are highly effective Pickering emulsifiers [37, 38]. For example, Madivala et al. found that the emulsion droplet stability depended strongly on the particle aspect ratio when using elongated hematite or polystyrene latex particles . Similarly, a recent study by Kalashnikova et al. reported the use of cellulose nanorods to form Pickering emulsions. Interestingly, it was found that too high an aspect ratio enabled these particles to adsorb simultaneously onto multiple droplets, rather than stabilizing individual droplets .
The recent development of living radical polymerization techniques [41, 42, 43, 44, 45, 46] has provided a facile route for the production of diblock copolymer nanoparticles based on polymerization-induced self-assembly (PISA) [47, 48, 49, 50, 51, 52, 53, 54]. In particular, the ability to form well-defined amphiphilic diblock copolymers via reversible addition-fragmentation chain transfer (RAFT) polymerization under appropriate reaction conditions allows the in situ formation of copolymer nanoparticles with either spherical, worm-like, or vesicular morphologies [52, 55, 56, 57, 58, 59]. Moreover, this approach has proven to be highly versatile, with all three copolymer morphologies being reported as pure phases in either water [52, 60, 61], alcohol [55, 58, 62, 63, 64, 65], or n-alkanes [59, 66, 67]. Aqueous emulsion polymerization has been extensively researched by Charleux and coworkers [50, 68, 69, 70, 71, 72, 73, 74]. However, RAFT dispersion polymerization formulations are arguably rather more versatile [58, 59, 61, 62, 64, 65, 75, 76, 77, 78, 79, 80]. Of particular relevance to the present study, Thompson et al. have recently shown that block copolymer worms prepared via RAFT PISA are effective stabilizers for non-aqueous emulsions comprising ethylene glycol droplets within various n-alkanes . Herein, we utilize a RAFT PISA formulation to conveniently prepare diblock copolymer worms directly in methanol. These flexible, highly anisotropic nanoparticles are then examined as putative Pickering emulsifiers for the preparation of new non-aqueous emulsions composed of sunflower oil droplets dispersed in a methanolic continuous phase. Thus, this new non-aqueous emulsion formulation is complementary to that reported by Thompson et al. .
Materials and methods
Ethanol was obtained from VWR Chemicals (UK) and n-hexane was purchased from Fisher Scientific (UK). All other reagents were purchased from Sigma-Aldrich (UK) and were used as received unless otherwise noted. Either 4,4′-azobis(4-cyanovaleric acid) (ACVA) or 2,2′-azobis(isobutyronitrile) (AIBN) were used as initiators. Benzyl methacrylate (BzMA) (96 %; Sigma-Aldrich) was passed through an inhibitor removal column prior to use. 4-Cyano-4-(2-phenyl-ethanesulfanylthiocarbonyl) sulfanylpentanoic acid (PETTC) was prepared in-house as reported previously .
Synthesis of poly(2-(dimethylamino)ethyl methacrylate) (PDMA) macro-CTA agent
A round-bottomed flask was charged with 2-(dimethylamino)ethyl methacrylate (DMA; 40.0 g, 254 mmol), PETTC (2.156 g, 6.36 mmol; target DP = 40), ACVA (178 mg, 0.636 mmol; PETTC/ACVA molar ratio = 10) and THF (40.0 g). The sealed reaction vessel was purged with nitrogen and placed in a pre-heated oil bath at 66 °C for 6 h. The resulting crude PDMA (monomer conversion = 77 %; Mn = 6500 g mol−1, Mw/Mn = 1.22) was purified by precipitation into excess petroleum ether. The mean degree of polymerization (DP) of this PDMA macro-CTA was calculated to be 43 using 1H NMR spectroscopy by comparing the integrated signals corresponding to the aromatic protons at 7.2–7.4 ppm with those assigned to the methacrylic polymer backbone at 0.4–2.5 ppm.
Synthesis of poly(2-(dimethylamino)ethyl methacrylate)-poly(benzyl methacrylate) (PDMA-PBzMA) diblock copolymer particles via dispersion polymerization in methanol
In a typical RAFT dispersion polymerization synthesis conducted at 15 % w/w total solids, BzMA (2.0 g, 11.4 mmol), PDMA43 macro-CTA (0.85 g, 0.119 mmol; target DP = 95) and AIBN (3.9 mg, 0.024 mmol; macro-CTA/AIBN molar ratio = 5) were dissolved in methanol (16.16 g). The reaction mixture was sealed in a round-bottomed flask, purged with nitrogen gas for 15 min, and then placed in a preheated oil bath at 64 °C for 24 h. The final monomer conversion was determined by 1H NMR analysis by comparing the integral due to the two benzylic protons assigned to the PBzMA block at 4.9 ppm to that of the BzMA monomer vinyl signals at 5.2 and 5.4 ppm.
Diblock copolymer molecular weight distributions were assessed using gel permeation chromatography (GPC). The GPC set-up comprised two 5 μm (30 cm) “Mixed C” columns and a WellChrom K-2301 refractive index detector operating at 950 ± 30 nm. THF eluent contained 2.0 % v/v triethylamine and 0.05 % w/v butylhydroxytoluene (BHT) was used at a flow rate of 1.0 ml min−1. A series of ten near-monodisperse linear poly(methyl methacrylate) standards (Mp ranging from 1280 to 330,000 g mol−1) were purchased from Polymer Laboratories (Church Stretton, UK) and employed for calibration using the above refractive index detector.
1H NMR spectra were acquired on a Bruker 400 MHz spectrometer in either CDCl3 or CD2Cl2. All chemical shifts are reported in ppm (δ). DLS measurements were conducted using a Malvern Instruments Zetasizer Nano series instrument equipped with a 4 mW He-Ne laser operating at 633 nm, an avalanche photodiode with high quantum efficiency, and an ALV/LSE-5003 multiple tau digital correlator electronics system.
TEM studies were conducted using a Philips CM 100 instrument operating at 100 kV equipped with a Gatan 1 k CCD camera. Solutions were diluted with methanol at 20 °C to generate 0.20 % w/w dispersions. Copper/palladium TEM grids (Agar Scientific, UK) were surface-coated in-house to yield a thin film of amorphous carbon. The grids were then plasma glow-discharged for 30 s to create a hydrophilic surface. Each methanolic diblock copolymer dispersion (0.20 % w/w, 10 μl) was placed onto a freshly glow-discharged grid for 1 min and then blotted with filter paper to remove excess solution. To stain the deposited nanoparticles, 10 μl of a 0.75 % w/w aqueous solution of uranyl formate was placed on the sample-loaded grid via micropipet for 20 s and then carefully blotted to remove excess stain. Each grid was then carefully dried using a vacuum hose.
Preparation of Pickering emulsions
Sunflower oil (5.0 ml) was homogenized with 5.0 ml of a 0.01–2.65 % w/w methanol copolymer dispersion for 2 min at 20 °C using a IKA Ultra-Turrax T-18 homogenizer equipped with a 10 mm dispersing tool operating at between 3500 and 13,500 rpm. The homogenizer was washed thoroughly using methanol between samples to ensure that there was no cross-contamination.
Optical microscopy images of Pickering emulsion droplets were recorded using a Motic DMBA300 digital biological microscope equipped with a built-in camera and analyzed using Motic Images Plus 2.0 ML software.
Each emulsion was sized in methanol using a Malvern Mastersizer 2000 instrument equipped with a small volume Hydro 2000SM sample dispersion unit (ca. 50 ml), a He-Ne laser operating at 633 nm and a solid-state blue laser operating at 466 nm. The stirring rate was adjusted to 500 rpm in order to avoid droplet coalescence. After each measurement, the cell was rinsed once with doubly distilled water, followed by rinsing with first ethanol and then methanol. The glass walls of the cell were carefully wiped with lens cleaning tissue to avoid cross-contamination and the laser was aligned centrally to the detector prior to data acquisition.
Determination of Pickering emulsifier adsorption efficiency via turbidimetry
UV spectra were recorded at 20 °C for the PDMA43-PBzMA94 worms in methanol using a Shimadzu UV-1800 instrument operating between 400 and 800 nm. A linear calibration plot of absorbance versus concentration at an arbitrary wavelength of 430 nm with known concentrations of copolymer dispersed in methanol was constructed in order to determine the nanoparticle adsorption efficiency at the oil-ethanol interface. This was assessed by analysis of the (upper) methanol continuous phase after sedimentation of the relatively dense sunflower oil droplets had occurred on standing for 24 h (or longer) at 20 °C. The remaining non-adsorbed worms were detected and thus the fraction of adsorbed worms was calculated by difference.
Results and discussion
Attempted Pickering emulsification of various oils using a methanolic dispersion containing 0.66 % w/w PDMA43-PBzMA94 diblock copolymer worms. Homogenization conditions: 13,500 rpm for 2 min at 20 °C using a sunflower oil volume fraction of 0.50
Initial emulsion, but demulsified after 2–3 h
Complete phase separation
Complete phase separation
Complete phase separation
Miscible with methanol
Effect of varying the PDMA43-PBzMA94 worm concentration on the mean droplet diameter, fractional surface coverage (Cw) and the adsorption efficiency of the worms on the sunflower oil droplets
Initial emulsion after 24 h
Aged emulsion after 7 days
Aged emulsions after 2 months
Mean laser diffraction droplet diameter (μm)
Pickering emulsion adsorption efficiency (%)
Mean laser diffraction droplet diameter (μm)
Pickering emulsion adsorption efficiency (%)
Mean laser diffraction droplet diameter (μm)
1.32 % w/w
9 ± 5
37 ± 30
40 ± 32
0.66 % w/w
9 ± 6
39 ± 21
39 ± 16
0.33 % w/w
14 ± 7
43 ± 18
44 ± 17
0.04 % w/w
48 ± 28
77 ± 31
79 ± 29
0.02 % w/w
53 ± 28
116 ± 60
104 ± 67
In principle, an alternative mechanism for the observed increase in emulsion size could be Ostwald ripening. According to Weidner and coworkers, the solubility of sunflower oil in methanol is approximately 0.5–1.0 % w/w at 20 °C . Thus, Ostwald ripening might occur for the present Pickering emulsion formulation via gradual diffusion of the sparingly soluble sunflower oil from smaller to larger droplets. However, this explanation does not appear to be consistent with the experimental observations. Interfacial adsorption of the worms is expected to be strong and essentially irreversible. Thus, if such sunflower oil diffusion occurred, both an increase in mean droplet diameter and a reduction in Cw would be expected. In practice, only the former change is observed—the worm surface coverage actually increases as the emulsion coarsens on ageing. In summary, we suggest that the increase in emulsion droplet dimensions over time is most likely the result of a limited coalescence mechanism. However, one reviewer of this manuscript has suggested that our experimental observations may be consistent with Ostwald ripening, provided that the worms released after preferential dissolution of the smaller droplets are partially readsorbed onto the growing larger droplets. It seems that further studies are warranted to clarify the true situation, but unfortunately, this is beyond the scope of the present study.
Sunflower oil-in-methanol Pickering emulsions can be prepared using PDMA43-PBzMA94 diblock copolymer worms as Pickering emulsifiers. Increasing the particle concentration allows stabilization of smaller droplets (<10 μm at 1.32 % w/w) but emulsions formed at particle concentrations as low as 0.02 % w/w also remained stable (average droplet diameter 53 μm). Systematically varying the stirring speed during homogenization produced emulsions with adjustable diameters up to a sunflower oil volume fraction of 0.60. Turbidimetry studies were employed to assess Pickering adsorption efficiency and an appreciable increase in mean droplet diameter was observed on ageing at ambient temperature. On closer inspection, no further increase in droplet diameter occurred after around 4 days. At all worm concentrations investigated, Pickering adsorption efficiencies were lower for 7-day-old emulsions than for the initial emulsion, while the worm fractional surface coverage increased significantly on this time scale. After this ageing period, the droplet diameter remained essentially unchanged for at least 2 months. Based on these experimental observations, we suggest that this increase in droplet diameter is the result of limited coalescence. Methanol-in-sunflower oil-in-methanol double emulsion droplets were also observed for this non-aqueous Pickering emulsion formulation and this observation warrants further studies.
We thank DSM Advanced Surfaces (Geleen, The Netherlands) and EPSRC for a CASE PhD studentship for ERJ. SPA thanks EPSRC (platform grant EP/J007846/1) for post-doctoral support of KLT and also acknowledges partial project funding from Stichting Innovatie Alliantie (Foundation Innovation Alliance).
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