Pickering and Network Stabilization of Biocompatible Emulsions Using

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Pickering and network stabilization of biocompatible emulsions using chitosan-modified silica nanoparticles Lauriane Alison, Patrick Alberto Rühs, Elena Tervoort, Alexandra Teleki, Michele Zanini, Lucio Isa, and André R. Studart Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b03439 • Publication Date (Web): 25 Nov 2016 Downloaded from http://pubs.acs.org on November 27, 2016

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Pickering and network stabilization of biocompatible emulsions using chitosanmodified silica nanoparticles Lauriane Alison*, Patrick A. Rühs*, Elena Tervoort*, Alexandra Teleki§, Michele Zanini†, Lucio Isa†, André R. Studart* * Complex Materials, Department of Materials, ETH Zü rich, 8093 Zü rich, Switzerland. § DSM Nutritional Products Ltd., Nutrition R&D Center Formulation and Application, P.O. Box 2676, 4002 Basel, Switzerland. † Interfaces, Soft Matter and Assembly, Department of Materials, ETH Zü rich, 8093 Zü rich, Switzerland. Corresponding author: André R. Studart [email protected]

Abstract Edible solid particles constitute an attractive alternative to surfactants as stabilizers of food-grade emulsions for products requiring a long-term shelf life. Here, we report on a new approach to stabilize edible emulsions using silica nanoparticles modified by noncovalently bound chitosan oligomers. Electrostatic modification with chitosan increases the hydrophobicity of the silica nanoparticles and favors their adsorption at the oilwater interface. The interfacial adsorption of the chitosan-modified silica particles enables the preparation of oil-in-water emulsions with small droplet sizes of a few microns through high-pressure homogenization. This approach enables the stabilization of food-grade emulsions for more than three months. The emulsion structure and stability can be effectively tuned by controlling the extent of chitosan adsorption on the silica particles. Bulk and interfacial rheology are used to highlight the two stabilization mechanisms involved. Low chitosan concentration (1wt% with respect to silica) leads to the formation of a viscoelastic film of particles adsorbed at the oil-water interface, enabling Pickering stabilization of the emulsion. By contrast, a network of agglomerated particles formed around the droplets is the predominant stabilization mechanism of the emulsions at higher chitosan content (5wt% with respect to silica). These two pathways

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against droplet coalescence and coarsening open up different possibilities to engineer the long-term stabilization of emulsions for food applications.

Introduction Many food products utilize emulsions either as an intermediate soft template during processing or as a final formulation for the distribution and consumption of nutritional ingredients. The properties and stability of such emulsions depend on the chemical nature and the ratio of the two constituent immiscible fluids, the droplet size distribution and the stabilizer used at the liquid-liquid interface. Emulsion destabilization needs to be avoided during food processing but also over the shelf life of the products to prevent undesirable sensorial and visual aspects. In applications that require the design and delivery of fat-soluble compounds such as carotenoids, vitamins and poly unsaturated fatty acids, long-term stable oil-in-water emulsions are essential to obtain the desired ingredients in the final formulation. Among those ingredients, carotenoids represent a widely used family of additives in the food industry mainly used for coloration, fortification and as nutritional supplements.1 For the formulation of beverages, the encapsulation of active food ingredients such carotenoids or fat-soluble vitamins requires the development of stable oil-in-water emulsions with Brownian droplets of a few microns in size. However, some food-grade emulsions still suffer from limited stability against droplet coalescence and Ostwald ripening, as visible in food products through phase separations, color changes or presence of sediments. To reduce such limited stability, surfactants, hydrocolloids or proteins are generally used in the food industry as emulsion stabilizers. Proteins adsorb at the oil-water interface and stabilize emulsions through the formation of a protective viscoelastic layer around the droplets. By rearranging their chain configuration in the form of “loops and trains“ in such protective layers, proteins lead to repulsive forces between droplets that help prevent coalescence.2 For example, gelatin is a protein commonly used as a stabilizer in carotenoid formulations.3 In another approach, the favorable interactions between proteins and polysaccharides can also be harnessed for the encapsulation of nutrients by forming interfacial complexes on the droplet surface.4 Water-insoluble proteins such as corn protein zein have also been recently utilized as effective stabilizers for oil-in-


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water emulsions.5 Although commonly used for food emulsions, proteins might not provide the long-term stability required in some applications.6 Furthermore, current formulations based on gelatin are still not fully satisfactory and need to be replaced to fulfill regulatory agencies, consumer perception, and dietary demands. Attempts to replace animal-based gelatin with gum arabic7 and modified food-starch8-9 have been made, but the long-term stabilities achieved in those systems are not comparable to those of gelatin-containing emulsions.10 Moreover, food starch is generally chemically modified when it is used as a stabilizer for oil-in-water emulsions. This is a major drawback as only a certain amount of modification is allowed by regulation. To address these issues, it would be highly desirable to use solid particles that could stabilize droplets in the form of Pickering emulsions. In spite of their typically larger droplet size compared to systems formulated with proteins and small weight surfactants, Pickering emulsions exhibit enhanced long-term stability. This stems from the fact that the energy required to remove a particle from the interface is orders of magnitude higher than that needed to desorb surfactants.11 Due to their high energy of attachment, particles are practically irreversibly anchored at the interface, forming layers that provide a mechanical barrier against coalescence and Ostwald ripening.12 The stabilization of food-grade emulsions with particles has been proposed in the literature but remains challenging for two reasons. First, the availability of edible surface-active particles is limited due to restricting food regulations. Second, it is difficult to generate micron- or even sub-micron sized Pickering-stabilized droplets, which is the typical droplet size needed to improve appearance and stability of many food products.13-14 Recently, particles of biological origin have been efficiently used to stabilize Pickering emulsions.15 While edible particles such as chitin nanocrystals16, cellulose17, wax18, and flavonoids19 lead to stable droplets, the average droplet sizes lie above 5 µm, which might be related to the low energy used for emulsification or by the possible lower stability provided by these systems. A potential candidate that is edible and could generate smaller droplets are silica nanoparticles. Silica is commonly used in food products as a flow aid agent or as a stabilizer, for example, for water-in-oil emulsions in chocolate confectionary.20 To be used as emulsion stabilizers, the surface activity of silica nanoparticles has to be tuned to obtain an appropriate degree of hydrophobicity that ensures optimum wettability at the oil-water interface. Surface modification strategies that enable such hydrophobicity


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control range from chemical grafting to physical adsorption of specific molecules onto the particle surface.21 According to current regulatory guidelines, covalently modified particles have to be treated as a new compound and thus would not be covered by existing food regulations.22-23 An attractive alternative is to modify silica nanoparticles non-covalently by adsorbing, for example, low-molecular weight charged amphiphilic molecules on the particle surface.24 To create edible particles, such strategy should utilize only food-grade building blocks.22-23 A good candidate for such building blocks is chitosan, an edible polysaccharide with a hydrophobic backbone and positively charged amino groups obtained at pHs below its pKa value. Chitosan-based emulsifiers have been shown to be effective stabilizers for Pickering emulsions due to electrostatic interactions between the molecule’s cationic groups and negatively charged moieties on particles or bacterial cells. Nonetheless, the resulting particle-stabilized droplets are typically larger than 20 µm.25-27 Additionally, water-in-oil emulsions have also been successfully stabilized through the physical adsorption of chitosan on silica under acidic conditions.20 However, due to the large fractal-like aggregate structure of the fumed silica particles utilized in such systems, the final emulsion droplets obtained were larger than 10 µm. As micron-sized droplets are essential in many food applications, it is highly desirable to identify processing routes that can decrease the droplet size of Pickering emulsions while using an edible particlebased stabilizer. Here, we show that chitosan-modified silica nanoparticles can be utilized to generate particle-stabilized emulsions with 1 µm-sized oil-in-water droplets that resist destabilization for more than three months. A non-covalent approach is used to coat the surface of silica nanoparticles with water-dispersible chitosan oligomers, thus creating modified particles that can readily adsorb on the droplet surface or form a percolating network in the continuous phase. We first investigate the electrostatic adsorption of positively charged chitosan on the surface of non-agglomerated negatively charged silica nanoparticles. By studying the influence of the chitosan concentration on the surface properties of silica nanoparticles and on the rheology of the resulting suspensions, we establish the conditions required to control the morphology and stability of oil-in-water emulsions generated in a high-pressure homogenizer. Confocal microscopy, cryo-scanning electron microscopy, diffusing wave spectroscopy, bulk and interfacial rheology are then used to shed light on the mechanisms underlying the high


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emulsion stability. Through such a systematic study, we developed a novel pathway to create long-term stable food-grade emulsions using chitosan-modified silica nanoparticles as edible building blocks.

Experimental section Materials Chitosan oligosaccharide dispersible in water between pH 3 and 7 was supplied by Haide Bei (China). This chitosan grade exhibits a degree of deacetylation higher than 85%, molecular weight MW ≤3 kDa and pKa of 6.5. Corn oil (specific gravity, ρ=0.9 g.mL1)

and silica Ludox TM50 suspensions were purchased from Sigma-Aldrich (Germany)

and used without any purification. The Ludox TM50 suspensions consisted of 50wt% of 22nm silica nanoparticles in water at a pH of 9 (suspension density, ρ=1.4 g.mL-1). MilliQ water with an electrical resistivity of 18.2 mΩ.cm was used to prepare a chitosan solution and to dilute the commercial particle suspension.

Preparation of chitosan-modified silica suspensions The chitosan solution was prepared at a concentration of 20wt% by adding drop-wise the chitosan solid powder into Milli-Q water under stirring until total dissolution. Although the chitosan is found to be slightly agglomerated in this initial solution, we have observed that the high shear forces produced during the emulsification process de-agglomerate the chitosan oligomers in water. The pH value of the chitosan solution was approximately 6. Chitosan-modified suspensions were obtained by diluting the Ludox TM50 suspension with Milli-Q water, and then adding drop-wise the chitosan solution under magnetic stirring. Unless specified otherwise, silica suspensions were prepared at a concentration of 8.7wtsusp%. Preliminary experiments in emulsions containing silica concentrations in the range 0.5–8.7% suggest that a particle content of 8.7wt% is sufficiently high to enable the stabilization of small droplets and to avoid macroscopic phase separation through the formation of a particle network in the continuous phase. Here, wtsusp% denotes the concentration calculated with respect to the total weight of the suspensions. The concentrations of chitosan were calculated with respect to the silica content (wtSiO2%). To improve readability, chitosan concentrations


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(wtSiO2%) will be denoted simply by wt%. The mixture was stirred at 1000 rpm for 15 minutes. The pH was adjusted to 5.5 using a 1M HCl solution prepared from Titrisol concentrates from Merck Millipore.

Preparation of Pickering emulsions stabilized by chitosan-modified silica nanoparticles Emulsions were prepared with 10wt% of corn oil and 90wt% of aqueous chitosanmodified silica suspensions. The mixture was mixed by hand before the preemulsification step performed with an Ultra-Turrax rotor-stator mixer (disperser T25 digital, dispersing tool S 25 N – 18 G from IKA, Germany) using a dispersing head operating at 10200 rpm for 1 minute. Emulsification was realized with a high-pressure homogenizer (Nano DeBEE Laboratory Homogenizer from BEE international, US) using a diamond nozzle (D5, orifice 150 µm). The suspension was processed four times, three times at 1380 bar and the last one at 2760 bar. To investigate the long-term stability of the Pickering emulsions, they were stored in a fridge at 4°C for several months.

Zeta potential measurements The chitosan-modified silica suspensions were titrated and the corresponding zeta potentials were recorded as a function of pH. The zeta potential of the chitosanmodified silica suspensions were measured using the electroacoustic Colloidal Vibration Current technique in a DT300 equipment at 25°C (Dispersion Technology, US). The titrations during the zeta potential measurements were performed from high to low pH using 1M HCl solution. The starting pH of each zeta potential curve corresponds to the initial pH of the modified silica suspensions (Figure 2a). This pH decreases with increasing the chitosan content due to the presence of more positively charged amino groups from the chitosan molecules, which behave as a weak acid and lower the pH by releasing protons. This oligomer is a chitosan chloride; some amino groups are always protonated. Because chitosan acts as a buffer, the zeta potential curve for the pure chitosan was obtained from solutions prepared at different pHs without using the autotitration function.

Droplet size characterization


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The oil droplets were investigated under a DM IL LED microscope (Leica, Germany) illuminated by an external UV light source (stereo microscope fluorescence adapter, Nightsea, US). Light emission of corn oil under UV excitation improved droplet visualization. The droplet size distribution of the emulsions was evaluated using a Mastersizer 2000 particle size analyzer based on laser diffraction (Malvern Instrument, UK). Mie theory was applied in the analysis using the following optical properties for the oil droplets: a refractive index of 1.5 and an absorption coefficient of 0.005. The transient droplet size distribution was monitored after emulsification (called “initial time”), 3 weeks and 3 months.

Adsorption experiments by total organic carbon (TOC) analysis Adsorption isotherm measurements were conducted using a total organic carbon analyzer (TOC VCSN, Shimadzu, Japan), which provided a quantitative measure of the non-adsorbed fraction of chitosan in the modified suspensions (laboratory of Génie Chimique, UMR5503 CNRS-INPT-UPS). 8.7wtsusp% silica suspensions coated with different concentrations of chitosan from 0 to 30wt% were prepared at pH 5.5. The suspensions were centrifuged at 20000 rpm for 2 h at 20°C and the supernatant aliquots were analyzed.

Rheological measurements The rheological properties of the suspensions and emulsions were measured with bulk experiments using a rheometer (Physica MCR 502, Anton Paar, Austria) with a double gap geometry (DG 26.7) at 25°C. The network properties were characterized with amplitude sweeps at a constant frequency of ω = 1 rad.s-1. The amplitude sweeps were performed by ramping the applied strain within the ranges 0.01-100% and 0.001-100% for the suspensions and emulsions, respectively. To measure the build-up and properties of the interfacial adsorption layer, interfacial shear rheology measurements were performed at 25°C with a rheometer (Physica MCR 501, Anton Paar, Austria) using a biconal geometry (BI-C68-5), which was placed at the oil-water interface.28 The Boussinesq number Bo was calculated to be greater than 1 allowing the interfacial flow to be decoupled from the bulk phase flow. The structure of the adsorption layer formed between the lower aqueous phase (chitosan-modified silica


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suspensions) and the oil phase (corn oil) was probed with a time sweep. Time sweeps were performed at a strain of γ = 0.01% and an angular frequency of ω = 0.3 rad.s-1 for 24 h.

Diffusing wave spectroscopy measurements Due to the high particle concentrations, the turbidity, and the polydispersity of the samples, diffusing wave spectroscopy (DWS) was selected to characterize the modified suspensions. DWS measurements were performed at 25°C in a DWS apparatus (LS instruments, Switzerland). The laser used as a light source had a wavelength of 683 nm and the backscattered light was collected on a photomultiplier. The samples were introduced in glass cuvettes having an optical path length of 1 cm. The intensity fluctuations were measured by a digital correlator and the electric field autocorrelation function g2(τ)-1 was determined.

Cryo-scanning electron microscopy To determine the arrangement of particles at the oil-water interface, freeze-fracture technique coupled with cryo-SEM was used.29 In short, 4 µL of the desired Pickering emulsion were sandwiched between two pre-cleaned and hydrophilized custom-made copper holders. The sample was then shock-frozen in a propane-jet freezer (BalTec/Leica JFD 030, Balzers/Vienna). The high cooling rate prevented water crystallization and fixed the position of the emulsion’s components as they were in the liquid state. The frozen samples were subsequently fractured under high vacuum conditions in a pre-cooled freeze-fracture device at -120°C (Bal-Tec/Leica BAF060 device). Samples were freeze-dried for 1 minute at -100 °C and then coated with 3 nm tungsten at a deposition angle of 30° followed by additional 3 nm at continuously varying angle between 30°and 90°. In this way, samples were uniformly coated and high magnifications could be easily achieved. Freeze-fractured metal-coated samples were then transferred to a pre-cooled SEM (-120°C) (Zeiss Gemini 1530, Oberkochen) for imaging.

Confocal microscopy analysis Emulsion droplets were observed with a Leica SP2 laser scanning confocal microscope


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(Leica Microsystems Inc., Germany). The oil phase was labeled with Nile Red (abcr, Germany). The stained emulsions were placed inside a glass capillary (0.1 x 1 mm) and sealed with epoxy adhesive and glued on a microscope slide (76 x 26 mm). Samples were observed with a 63x oil immersion lens (oil refractive index of 1.515) and by using a solid-state laser with wavelength of 561 nm. In order to visualize droplet motion qualitatively, two images with a time lag difference of 5 s were subtracted. Photobleaching was partially compensated via rescaling of the second image by the ratio of the two images’ mean intensities. When droplets are kept in place by a network, image subtraction leads to a uniform mask image with low standard deviation of the pixel-bypixel intensity variations. Otherwise, mobile droplets lead to larger intensity variations, and thus intensity standard deviations, in the subtracted image. Thermal drift was in all cases negligible. For each chitosan concentration, the motion of a representative oil droplet was tracked over 99 s with a frame rate of 1.01 FPS using Image J by comparing the coordinates found in each pair of two successive frames from a time series of xyimages obtained by confocal microscopy 3 days after emulsification.

Results and discussion Modification of silica nanoparticles with chitosan

The method proposed in this study relies on the in-situ non-covalent modification of particles to generate stable emulsions. Hydrophilic silica nanoparticles are surface-covered with modified chitosan (Figure 1a) to obtain partially hydrophobic particles that promote emulsion stabilization by adsorbing at oil-water interfaces. The hydrophobic character of the chitosan arises from its hydrocarbon backbone. Adsorption on the particles occurs mainly due to electrostatic interactions between the negatively charged silica and the positively charged chitosan oligosaccharides (Figure 1b). The charges along the chitosan molecules arise from the protonation of amine groups. Particle modification is accomplished at a pH close to the pKa value of the chitosan, since this promotes extensive ligand exchange interactions between the adsorbing ligands and the functional hydroxyl groups on the particle surface, as shown in earlier work.30-31 Electrostatic adsorption is accompanied by entropically favored release of counterions into the solution, which also promotes the


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formation of multilayers.32-34 In addition to amines, the water-dispersible chitosan chloride used in this study also contains protonated amine, acetyl, and glutamine moieties. Hydrogen bonds between acetyl groups of the chitosan molecules and hydroxyl groups on the silica surface have been suggested to provide an additional driving force for adsorption.30,35 In order to control the wettability of the particles at the oil-water interface, the chitosan concentration is adjusted to find the suitable balance between hydrophilicity and hydrophobicity on the silica surface. The surface-active modified particles are later used to stabilize oil droplets in an aqueous continuous phase (Figure 1c).

Figure 1: Schematic illustration of the formation of ultra-stable food-grade Pickering emulsions. (a) Hydrophobization of negatively charged silica nanoparticles by adsorption of chitosan through its positively charged amino groups. (b) Modification of silica nanoparticles by water-dispersible chitosan and pH adjustment to 5.5, close to the pKa value of chitosan. Gln stands for a glutamine group. (c) Stabilization of oil droplets through the adsorption of the chitosanmodified silica particles at the oil-water interface.

Characterization of chitosan-modified silica particles

Silica

particles modified with

electrostatically adsorbed chitosan are

characterized through zeta potential measurements and total organic carbon (TOC) analysis.


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The adsorption of chitosan on the particles is evaluated by surface charge measurements as a function of the chitosan concentration and pH (Figure 2a). Pure silica particles in water are negatively charged at the studied pHs, because the hydroxyl groups on their surface are deprotonated. By contrast, chitosan is positively charged within its pH range of solubility. Both components combined lead to chitosan-coated silica particles that display intermediate charges over the studied pH range. The charge of the coated particles becomes gradually more positive for increasing chitosan concentrations. For chitosan concentrations between 0.2wt% and 1wt%, the zeta potentials are similar to those of unmodified silica, indicating that silica particles are barely modified. At concentrations higher than 2.5wt%, the higher adsorption of chitosan leads to zeta potential values remarkably different compared to that of bare silica, reversing the charge at the particle surface from negative to positive. Increasing the chitosan content further to 10wt% and 30wt% leads to zeta potentials that are positive over the entire pH range studied, indicating that the silica particles are highly coated with chitosan. Indeed, at such high concentrations, multilayers are favored by hydrophobic interactions and by the release of counterions upon chitosan adsorption.30 The peaks in the zeta potential curves around a pH of 6.5 reveal that the interactions of the chitosan molecules with the silica surface are favored at the pKa value of the oligosaccharide. This effect is confirmed in the literature31 and becomes even more pronounced at higher chitosan concentrations. The decrease of zeta potential below the pKa value reflects the decrease of chitosan adsorption due to less negatively charged silica particles at low pH. This behavior is typical for weakly charged polyelectrolytes, where the main driving force for adsorption is electrostatic attraction.30 For this study, pH 5.5 is chosen as an optimum value for all studied concentrations.

To directly quantify the amount of chitosan adsorbed on the silica particles, we perform TOC analysis of the supernatant of the chitosan-modified silica suspensions after centrifugation. The analysis assumes that the precipitate contains chitosanmodified silica nanoparticles and the supernatant free chitosan (non-adsorbed chitosan). From the initial chitosan concentration added to the suspension and the carbon content measured in the supernatant, the method enables the determination of the amount of carbon in the precipitate and therefore the amount of adsorbed chitosan


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(Figure 2b). The amount of chitosan adsorbed on silica increases with the chitosan concentration initially used in the modified suspensions. In agreement with the zeta potential data (Figure 2a), the amount of molecules adsorbed increases continuously with chitosan content without reaching a plateau. For instance, particles in the suspension modified with 5wt% of chitosan contain an adsorbed content that is as much as three times higher than that of particles exposed to 1wt% of chitosan. Interestingly, a total adsorption of all the molecules present in the aqueous phase is only observed for a very low chitosan concentration below 1wt%. Beyond this concentration, the addition of chitosan inevitably leads to the presence of free chitosan in solution.

Figure 2: (a) Zeta potential measurements performed in 8.7wtsusp% silica suspensions containing different concentrations of chitosan. The modifier content is shown relative to the silica concentration. The results for two controls are shown for comparison: pure silica at a concentration of 8.7wtsusp% and pure chitosan at a concentration of 150ppm in aqueous solution. The lines are guides to the eye. (b) Adsorption isotherm of chitosan on silica nanoparticles obtained by total organic carbon (TOC) analysis. Values obtained from the organic carbon found in the precipitate after centrifugation of the 8.7wtsusp% silica suspensions modified with different chitosan concentrations at pH 5.5. The amount of adsorbed chitosan is plotted against the initial chitosan concentration (with respect to the silica content). The diagonal line represents the hypothetical case of 100% chitosan adsorption.

Agglomeration of chitosan-modified silica particles

Chitosan-modified silica particles agglomerate in water and form viscoelastic gels for modifier concentrations higher than 1wt%. Since a concentration window


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between 1 and 5wt% chitosan at pH 5.5 is found to be suitable for the stabilization of emulsions, we investigate the agglomeration behavior of such suspensions more closely through rheology, light scattering, and microscopic analysis. The zeta potentials at such conditions are low (Figure 2a), preventing electrostatic stabilization of the particles. As a result, particles form agglomerates through van der Waals and hydrophobic interactions36, whose presence in the continuous aqueous phase and at the oil-water interface favor the emulsion stability. Therefore, the colloidal behavior of the modified particles in water is likely dominated by hydrophobic interactions rather than electrostatic forces. Because 46% of the 5wt% of chitosan initially added to the suspension remains non-adsorbed in the aqueous medium (Figure 2b), the agglomeration process might also be favored by depletion flocculation. This tendency is confirmed with higher chitosan concentrations (see Figure S1). The agglomeration of particles has a direct effect on the rheological behavior of the bulk suspensions. Oscillatory rheological measurements (Figure 3a) reveal a viscoelastic flow behavior for both studied chitosan concentrations of 1 and 5wt%. As expected, much higher elastic (G’) and viscous moduli (G’’) are observed with a higher concentration of chitosan (5wt%) at low strain, in spite of the constant silica content. We hypothesize that such higher elastic modulus is related to the formation of stronger and larger agglomerates in the presence of increased quantities of surface modifier. Complementary rheological tests with 0.2wt% and 10wt% of chitosan-modified particles confirm this tendency (see Figure S2). The formation of agglomerates is further evaluated by diffusing wave spectroscopy (DWS, Figure 3b). The decay of the autocorrelation function occurs at relatively short time-scales for the suspension modified with 1wt% of modifier indicating restructuring and partial gravitational collapse of the gel network. However, the autocorrelation function for the suspension modified with 5wt% of chitosan does not decay to zero within the experimental time window due to the strong gel-like behavior of the suspensions. This indicates restricted particle motion in the presence of higher chitosan content, confirming the formation of a stronger network of particle agglomerates. This slower particle dynamics is associated with an increased viscoelasticity, which is in line with the results in Figure 3a. In addition, the time evolution of the autocorrelation functions for different chitosan-modified silica suspensions reveals that the decay is progressively shifted towards longer delay times


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after 1 day, indicating a further slowing down of the dynamics of the network structure (see Figure S1). This reveals that the gel-like behavior evolves and becomes more pronounced with time. To gain additional insights into the morphology of the agglomerates and their networks in the modified suspensions, cryo-SEM analysis is performed (Figure 3c). In agreement with the DWS measurements, larger agglomerates are observed in the presence of higher chitosan concentrations. With 5wt% chitosan thicker and more compact gel branches are found, whereas a more uniform structure with thinner gel strands is observed at 1wt% of the modifier. Combined, our results schematically represented in Figure 3d reveal that suspensions prepared with low chitosan concentrations contain smaller agglomerated flocs weakly bound together. In contrast, higher chitosan concentrations lead to higher attractive forces, thus resulting in a stronger viscoelastic network of larger particle agglomerates. According to the photographs of the modified silica suspensions, the gel at lower chitosan concentration partially collapsed with time under gravity, as opposed to the one prepared with the higher chitosan concentration, providing further evidence of the formation of a weaker network in the former case.


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Figure 3: (a) Storage G’ and loss G” moduli of silica suspensions modified with 1wt% or 5wt% of chitosan at time t0 (shortly after suspension preparation). (b) Normalized autocorrelation function at t0 obtained by diffusing wave spectroscopy (DWS) applied to silica suspensions modified with 1wt% or 5wt% of chitosan and the corresponding (c) cryo-SEM images. (d) Photographs taken after 7 months and schematic illustrations showing the agglomerates formed in silica suspensions modified with 1wt% (left) or 5wt% (right) of chitosan. All suspensions were prepared with 8.7wtsusp% of silica at pH 5.5.


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Emulsions stabilized by chitosan-modified silica particles

Mixing of the suspensions of chitosan-modified silica nanoparticles with corn oil using a high-pressure homogenizer leads to emulsions containing oil droplets of a few microns in a continuous aqueous phase (Figure 4a). We suggest that the particle agglomerates present in the initial suspension (Figure 3) are broken apart in the highpressure homogenization process, enabling efficient stabilization of the oil-in-water emulsions. The adsorption of modified particles on the surface of the droplets is confirmed by cryo-SEM (Figure 4b and c). While chitosan-modified silica particles can be used as efficient emulsion stabilizers, emulsions with only hydrophilic silica or chitosan do not lead to a stable emulsion. Comparative stability experiments at pH 5.5 confirm that, when used alone, neither silica nor chitosan are able to generate such stable emulsions (see Figure S3).

Figure 4: (a) Optical microscopy image of the emulsion taken immediately after emulsification (initial time) while illuminating the sample with UV light. (b) Cryo-SEM image of oil-in-water particle-stabilized emulsion. (c) Close up of an oil droplet revealing particles at the oil-water interface. Emulsions were prepared from 8.7wtsusp% silica suspensions modified with 5wt% of chitosan at pH 5.5. The oil-to-water ratio of the emulsion was 1:9.

To better understand the morphology and the internal microstructure of the obtained oil-in-water emulsions, confocal microscopy experiments and droplet tracking are conducted on four emulsions containing silica particles modified with 0.2wt%, 1wt%, 5wt%, and 10wt% of chitosan at pH 5.5. By increasing the chitosan content, the particles become more hydrophobic, tend to agglomerate faster and the concentration of free non-adsorbed molecules increases, which directly influences the emulsion stability. Photographs depicted in Figure 5i show the macroscopic behavior of the emulsions and the confocal images (Figure 5 ii) reveal the emulsion microstructure.


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Image subtraction and motion tracking of droplets are carried out to demonstrate the influence of the chitosan concentration on the real-time dynamics and structure of the emulsions (Figure 5 iii & iv and see Videos V1, V2, V3 and V4 in the Supporting Information). Together with the zeta potential, bulk rheology and diffusing wave spectroscopy measurements, the experimental macroscopic observations, confocal microscopy results and droplet mobility data (Figure 2a, Figure 3, Figure 5 i to iv) allow us to propose a model to describe the effect of chitosan concentration on the microstructure of the emulsion (Figure 5v). With 0.2wt% of chitosan (Figure 5a), the oil droplets on the confocal image are larger than with other chitosan concentrations due to an insufficient surface activity of the particles resulting in low adsorption at the interface and thus droplet coalescence. As the suspensions modified with 0.2wt% of chitosan do not gel and contain almost no agglomerates (Figure S1a), no accumulation of particles is expected at the oil-water interface. This is in agreement with the photograph of the corresponding emulsion, which shows creaming of the emulsion with this modifier concentration. Indeed, creaming occurs due to the absence of a network and the presence of separate flocs of droplets. The serum at the bottom of the sample appears as cloudy as the modified silica suspension indicating that a large number of the particles are still present in the aqueous phase. In this phase-separating system, the confocal microscopy video shows that the flocs of droplets are not stationary but highly mobile in the aqueous phase in spite of the observed creaming effect 37. The image resulting from the subtraction of two sequential snapshots presents a high value for the intensity standard deviation, indicating that the droplets are mobile with uncorrelated dynamics. The trajectories of two distinct droplets are tracked in Figure 5a(iv); one is part of a floc and rattles in a confined region, whereas the other is a free Brownian droplet that moves faster and independently from the other surrounding droplets. Droplet flocs are probably formed due to the low amount of chitosan on particles leading to a low concentration of almost non-agglomerated particles at the interface (see Figure S1a) and thus poor steric stabilization. The mobility of the resulting flocs correlates with the absence of a percolating network of particles throughout the continuous phase. The attraction between droplets is favored by the local high shear imposed to the system during the high-energy emulsification process. Alternatively, the low coverage of particles at the oil-water interface might also favor the adsorption of the same particle simultaneously


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onto different droplets, leading to the formation of flocs of droplets through a particlebridging effect.38 By increasing the initial chitosan concentration to 1wt% (Figure 5b), welldispersed small droplets are formed, as highlighted by the confocal image. The hydrophobization of silica particles is sufficient to increase their surface activity and therefore promote their extensive adsorption at the oil-water interface. This enables the formation of a protective coating of particles on the droplet surface, preventing coalescence upon shear-induced contact and leading to unflocculated emulsions. As previously discussed, agglomerates are formed in the suspensions and are likely to exist also at the oil-water interface for chitosan contents equal or higher than 1wt% (Figure 3). Droplet tracking shows motion over noticeable distances but within a confined space, indicating that the droplets are bound to a stationary network.

37

The value for

the intensity standard deviation between consecutive snapshots is intermediate in this case. Droplets move significantly less than in the system with 0.2wt% of chitosan and only rattle locally. The photograph confirms the weak gel-like behavior also observed for the modified silica suspensions (Figure 3 c & d). Indeed, the weaker attraction between the particles within agglomerates formed in the system with 1wt% chitosan leads to the collapse of the gelled-like emulsion and the expulsion of water out of the network (syneresis of the gel). The fact that droplets sediment over time indicates that they are now heavier than the continuous phase, confirming the adsorption of particles at the oil-water interface. The upper serum appears clean as most of the particles are trapped between the collapsed droplets. With further increase of chitosan up to 5wt% (Figure 5c), the droplets are still small and well-dispersed but the macroscopic behavior is different. No phase separation is observed, indicating that there is a space-filling particle network around the droplets that resists gravitational effects. For this chitosan concentration, homogeneous gelledlike emulsions are obtained since the agglomerates are stronger than in the formulation with 1wt% of chitosan (Figure 3). In contrast to the previous concentrations, the displacement of the dispersed droplets is restricted, suggesting the presence of a relatively strong network of particles within the continuous phase of the emulsion with 5wt% of chitosan. Indeed, the droplets are kept in position as illustrated by the resulting image after subtraction. These observations are in line with the effect of chitosan concentration on the agglomeration of particles in bulk suspensions (Figure 3).


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Because of extensive agglomeration within the continuous phase, a lower concentration of particles is expected to adsorb on the droplet surface, as compared to the high density of interfacially adsorbed particles predicted in emulsions with 1wt% chitosan. At a chitosan concentration beyond this optimum range (10wt%, Figure 5d), large flocs of droplets are observed in the confocal images, pointing to the existence of a heterogeneous network structure with even thicker branches and larger voids. Those flocs of droplets appear immobile in the droplet tracking and result in a subtracted image with low intensity standard deviation. Moreover, the photograph confirms the fact that the system is gelled, as no phase separation occurs even after 8 months. Based on the adsorption measurements shown in Figure 2b, half of the chitosan initially added to this emulsion is adsorbed on the silica particles whereas the rest is present as free chitosan in the bulk. The excess of free chitosan in solution leads to depletion attraction between the droplets and extensive agglomeration of particles within the aqueous phase due to hydrophobic interactions (see Figure S1d). This inhibits particle adsorption on the surface of the oil droplets24 and leads to the formation of an arrested particle network39 that traps droplets into fixed positions.


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Figure 5: Structure of oil-in-water emulsions prepared in the presence of chitosan-modified silica nanoparticles. (i) Photographs taken after 8 months of emulsions prepared with different amounts of chitosan (a) 0.2wt%, (b) 1wt%, (c) 5wt% and (d) 10wt%. (ii) Confocal images of those emulsions using fluorescent Nile red in the oil phase. (iii) Results of image subtraction with a time lag difference of 5 s to highlight the motion of the oil droplets. Larger standard deviation of the intensities indicates larger motion of the droplets. (iv) Trajectories of representative droplets over 100s performed for each chitosan concentration. (v) Schematic illustrations of the expected structure of particles at the oilwater interface and within the continuous aqueous phase depending on the chitosan concentrations used to prepare the emulsions. Emulsions were obtained from 8.7wtsusp% silica suspensions at pH 5.5. The oil-to-water ratio of the emulsion was 1:9.

Long-term stability of emulsions

As revealed by the confocal images displayed in Figure 5ii, silica nanoparticles are effective emulsion stabilizers when coated with 1wt% and 5wt% of chitosan. The long-term stability of the emulsions is investigated by evaluating the droplet size


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distribution over time for formulations containing different chitosan concentrations, using laser diffraction after dilution (Figure 6). With 0.2wt% of chitosan, the average droplet size (d50) is above 5 µm after emulsification (Figure 6a). Despite the low density of particles expected at the oil-water interface, the flocculated droplets do not grow with time. The lack of hydrophobicity of these particles was verified experimentally by an interfacial rheology measurement using a time sweep protocol over 20h (data not shown). Silica particles modified with 0.2wt% of chitosan at pH 5.5 do not spontaneously adsorb at the oil-water interface. We hypothesize that the high-energy involved during high-pressure emulsification lead to a minimum adsorption of particles at the interface, thus allowing for the stabilization of the droplets. Here, stability might be reached with a low surface coverage due to the presence of attractive interactions between the stabilizing particles40 and bridging particles that adsorb simultaneously at two different adjacent droplets. With higher chitosan concentration, particles agglomerate and form a network in the bulk. For 1wt% of chitosan, the emulsion is stable for three weeks with a d50 of 1.5 µm (Figure 6b). Nevertheless, the multimodal droplet size distribution of such emulsion after three months indicates destabilization and the appearance of larger droplets over time, which might result from Ostwald ripening at such long timescales. In the case of the formulation with 5wt% of chitosan, the emulsion is remarkably stable against coalescence and Ostwald ripening (Figure 6c). This composition leads to a narrow and monomodal droplet size distribution with an average droplet size of 1.7 µm that remains stable within a time period of up to three months after emulsification. Measurable changes are detected in the droplet size distribution of such emulsions only beyond four months (see Figure S4). The average size of the droplets measured by light scattering is in good agreement with the droplet size measured by optical microscopy (Figure 4a). By increasing the chitosan content further to 10wt%, a multimodal size distribution is already observed three weeks after emulsification (Figure 6d). The large scattering units measured in such multimodal distribution might correspond to droplets that have coalesced or to the flocs of droplets observed in the confocal images (Figure 5d), which could not be de-agglomerated by dilution due to hydrophobic interactions.


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Figure 6: Stability of emulsions assessed through the evolution of the droplet size distributions over time. Graphs at the left and right show size distributions in the form of discrete and cumulative volumes, respectively. The oil-in-water emulsions were prepared with 8.7wtsusp% silica suspensions modified with different amounts of chitosan at a pH of about 5.5: (a) 0.2wt%, pH 5.49; (b) 1wt%, pH 5.48; (c) 5wt%, pH 5,51; (d) 10wt%, pH 5.34. The oil-to-water ratio was 1:9.

Mechanisms of stabilization

Bulk and interfacial rheology measurements are performed to shed additional light onto the mechanisms governing the effective stabilization of emulsions containing nanoparticles modified with 1wt% and 5wt% chitosan. On the basis of these measurements, two different emulsion stabilization mechanisms are identified as discussed below (Figure 7). With a chitosan concentration of 1wt%, the emulsion is mainly stabilized by the modified particles adsorbed at the oil-water interface. Interfacial rheology reveals that in this case the adsorbed particles form a viscoelastic network at the interface, which becomes increasingly more elastic over time until it reaches constant values of G’i and G”i (Figure 7b). Despite the clear evidence of extensive particle adsorption at the oilwater interface, bulk rheological measurements show that the emulsion also exhibits a viscoelastic behavior (Figure 7a). Given that the droplets are not sufficiently concentrated to form a jammed elastic emulsion, such viscoelasticity suggests that a network is formed in this system and should also play a role in the emulsion stabilization. On the contrary, a chitosan concentration of 5wt% leads to an emulsion that is mainly stabilized by the formation of an attractive network of particles between the droplets. The presence of such strong network is confirmed by the bulk viscoelastic response of the suspension alone (Figure 3a), which is further amplified in the emulsion (Figure 7a). The storage and loss moduli (G’ and G”) are higher for the emulsion with 5wt% of chitosan, because attractive interactions increase with concentration, leading to a stronger network. Droplets in this emulsion are surrounded and connected to each other by the particle agglomerates described in Figure 3. For this system, no viscoelastic adsorption layer is detected by the interfacial rheology measurements (Figure 7b), suggesting that no network is formed at the interface. As the amount of chitosan adsorbed on the silica particles is three times higher than in the 1wt% system (Figure 2b), particles are expected to be very hydrophobic in suspensions containing 5wt%


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chitosan. However, some particles adsorb at the interface during the strong emulsification process (Figure 4 b & c). As a result, the strongly hydrophobized particles tend to readily agglomerate in the aqueous phase rather than adsorb on the surface of the droplets. The formation of a percolating network of agglomerated particles in the continuous phase is thus the main mechanism of stabilization of the emulsions containing 5wt% of chitosan. Although negatively charged oil droplets41 leads to electrostatic interactions that could also play an additional role on the adsorption of the modified particles at the oilwater interface, our results can be reasonably interpreted by assuming that the hydrophobic interactions between the chitosan-modified particles and between particles and the liquid interface are the main driving force controlling the morphology and stability of the investigated emulsions.

Figure 7: (a) Storage G’ and loss G” moduli of oil-in-water emulsions prepared from 8.7wtsusp% silica suspensions modified with 1wt% or 5wt% of chitosan at pH 5.5. Measurements were conducted directly after emulsification. (b) Storage Gi’ and loss Gi” moduli of the oil-water interface formed using corn oil as upper phase and 1wt% silica suspensions modified with 1wt% or 5wt% of chitosan at pH 5.5 as bottom phase. (c) Schematics of the main stabilization


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mechanisms proposed for emulsions containing different chitosan concentrations: Pickering stabilization for 1wt% of chitosan and network formation for 5wt% of chitosan.

The morphology of the particle network formed in the emulsion prepared with 5wt% chitosan is confirmed with cryo-SEM (Figure 8). Here, a high fraction of particles is found to be part of the percolating network around the oil droplets. During the emulsification process, such network is expected to break into flocs that either adsorb in small quantities at the oil-water interface or agglomerate extensively into a reconstituted network in the bulk phase over time. The viscoelastic nature of such network leads to the formation of a gelled emulsion, which resists destabilization processes like droplet coalescence and Ostwald ripening. Network stabilization has been previously reported in literature as an efficient approach to ensure long shelf life of emulsions thanks to its high mechanical stability and good resistance against creaming or sedimentation.42-43 That explains why the emulsion prepared with 5wt% of chitosan remains homogeneous over time and does not undergo any phase separation due to gravitational effects. Emulsions with such a high stability at pH 5.5 are interesting for food products such as milk or ice cream44 and also for soft drinks at lower pH. The highly concentrated emulsions studied in this work are meant to stabilize and deliver active ingredients like vitamins in food products and beverages. Thus, the emulsions would typically not be consumed as such and only constitute a small part of the final food product. In that context, the silica concentration in the final product will be low. Such a versatile system can be spray dried and redispersed in water at different pHs.

Figure 8: Cryo-SEM image of the percolating network of particles responsible for the stabilization of oil droplets in emulsions containing 5wt% of chitosan. The oil-in-water emulsion was prepared from 8.7wtsusp% silica suspension at a pH of 5.5. The oil-to-water ratio of the emulsion was 1:9.


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Conclusions Chitosan-modified silica particles can be effectively used as edible emulsifiers for the stabilization of biocompatible and food-grade emulsions. Micron-sized oil droplets that are stable for at least 3 months were successfully achieved by tailoring the interfacial adsorption

and

bulk

agglomeration

of

silica

nanoparticles

using

different

concentrations of partially hydrophobic chitosan molecules. Physical modification of silica nanoparticles is driven by electrostatic interactions between their negatively charged surface groups and the protonated water-dispersible chitosan at a pH around its pKa value. The oil-in-water emulsions prepared from such modified silica particles are stabilized through different mechanisms depending on the chitosan content used. With 1wt% of chitosan, modified silica particles are sufficiently hydrophobic to adsorb at the oil-water interface leading to stable Pickering emulsions. At the higher chitosan content of 5wt%, the particles become more hydrophobic and agglomerate extensively in the bulk to form a strong percolating network around the droplets. Through a highenergy emulsification process, such agglomerates are broken up and reformed around the oil droplets, leading to a network stabilized system. An effective combination of Pickering and network stabilization can possibly be explored in this system using adequate chitosan concentrations. Such emulsions can be used in the food, pharmaceutical or cosmetic industries for the encapsulation of active ingredients as a long-term stable alternative to conventional emulsions.

Acknowledgements We would like to thank DSM for the financial support (grant 3831/175101), Dr. Szilvia Mesaros and Dr. B.H. Leuenberger for fruitful discussions, Sreenath Bolisetty, Marion Frey, and Nadine Peneder for their contributions to the experimental part of this study, Sandrine Desclaux for the TOC analysis, and Ahmet Demirörs for his help with confocal microscopy and for valuable discussions. We are grateful to ScopeM microscopy center at ETH for providing access to their microscopes. Lucio Isa and Michele Zanini acknowledge financial support from the Swiss National Science Foundation (grant PP00P2_144646/1).


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36. Velev, O. D.; Furusawa, K.; Nagayama, K. Assembly of Latex Particles by Using Emulsion Droplets as Templates. 2. Ball-like and Composite Aggregates. Langmuir 1996, 12, 2385-2391. 37. ten Grotenhuis, E.; Paques, M.; van Aken, G. A. The Application of Diffusing-Wave Spectroscopy to Monitor the Phase Behavior of Emulsion–Polysaccharide Systems. Journal of Colloid and Interface Science 2000, 227, 495-504. 38. Horozov, T. S.; Binks, B. P. Particle-stabilized emulsions: A bilayer or a bridging monolayer? Angewandte Chemie - International Edition 2006, 45, 773-776. 39. Mezzenga, R.; Schurtenberger, P.; Burbidge, A.; Michel, M. Understanding foods as soft materials. Nat Mater 2005, 4, 729-740. 40. Arditty, S.; Whitby, C. P.; Binks, B. P.; Schmitt, V.; Leal-Calderon, F. Some general features of limited coalescence in solid-stabilized emulsions. Eur. Phys. J. E 2003, 11, 273-281. 41. Marinova, K. G.; Alargova, R. G.; Denkov, N. D.; Velev, O. D.; Petsev, D. N.; Ivanov, I. B.; Borwankar, R. P. Charging of Oil−Water Interfaces Due to Spontaneous Adsorption of Hydroxyl Ions. Langmuir 1996, 12, 2045-2051. 42. Ghosh, S.; Tran, T.; Rousseau, D. Comparison of Pickering and Network Stabilization in Water-in-Oil Emulsions. Langmuir 2011, 27, 6589-6597. 43. Lee, M. N.; Chan, H. K.; Mohraz, A. Characteristics of Pickering Emulsion Gels Formed by Droplet Bridging. Langmuir 2012, 28, 3085-3091. 44. Marshall R.T., D. G. H., Hartel R. W. Compositions and Properties. In Ice cream, sixth edition ed.; Springer US: New-York, 2003.

Supporting Information Diffusing wave spectroscopy and bulk rheology measurements of chitosan-modified silica suspensions. Control emulsions with only silica nanoparticles and only chitosan molecules as stabilizers. Droplet size distribution of emulsions showing long-term stability. This material is available free of charge via Internet at http://pubs.acs.org.


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Table of Contents Graphic

keywords: emulsions, Pickering emulsions, interfacial rheology, food emulsions, network stabilization, chitosan, and silica nanoparticles.


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Figure 1: Schematic illustration of the formation of ultra-stable food-grade Pickering emulsions. (a) Hydrophobization of negatively charged silica nanoparticles by adsorption of chitosan through its positively charged amino groups. (b) Modification of silica nanoparticles by water-dispersible chitosan and pH adjustment to 5.5, close to the pKa value of chitosan. Gln stands for a glutamine group. (c) Stabilization of oil droplets through the adsorption of the chitosan-modified silica particles at the oil-water interface.

160x100mm (300 x 300 DPI)


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Figure 2: (a) Zeta potential measurements performed in 8.7wtsusp% silica suspensions containing different concentrations of chitosan. The modifier content is shown relative to the silica concentration. The results for two controls are shown for comparison: pure silica at a concentration of 8.7wtsusp% and pure chitosan at a concentration of 150ppm in aqueous solution. The lines are guides to the eye. (b) Adsorption isotherm of chitosan on silica nanoparticles obtained by total organic carbon (TOC) analysis. Values obtained from the organic carbon found in the precipitate after centrifugation of the 8.7wtsusp% silica suspensions modified with different chitosan concentrations at pH 5.5. The amount of adsorbed chitosan is plotted against the initial chitosan concentration (with respect to the silica content). The diagonal line represents the hypothetical case of 100% chitosan adsorption.

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Figure 3: (a) Storage G’ and loss G” moduli of silica suspensions modified with 1wt% or 5wt% of chitosan at time t0 (shortly after suspension preparation). (b) Normalized autocorrelation function at t0 obtained by diffusing wave spectroscopy (DWS) applied to silica suspensions modified with 1wt% or 5wt% of chitosan and the corresponding (c) cryo-SEM images. (d) Photographs taken after 7 months and schematic illustrations showing the agglomerates formed in silica suspensions modified with 1wt% (left) or 5wt% (right) of chitosan. All suspensions were prepared with 8.7wtsusp% of silica at pH 5.5.

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Figure 4: (a) Optical microscopy image of the emulsion taken immediately after emulsification (initial time) while illuminating the sample with UV light. (b) Cryo-SEM image of oil-in-water particle-stabilized emulsion. (c) Close up of an oil droplet revealing particles at the oil-water interface. Emulsions were prepared from 8.7wtsusp% silica suspensions modified with 5wt% of chitosan at pH 5.5. The oil-to-water ratio of the emulsion was 1:9.

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Figure 5: Structure of oil-in-water emulsions prepared in the presence of chitosan-modified silica nanoparticles. (i) Photographs taken after 8 months of emulsions prepared with different amounts of chitosan (a) 0.2wt%, (b) 1wt%, (c) 5wt% and (d) 10wt%. (ii) Confocal images of those emulsions using fluorescent Nile red in the oil phase. (iii) Results of image subtraction with a time lag difference of 5 s to highlight the motion of the oil droplets. Larger standard deviation of the intensities indicates larger motion of the droplets. (iv) Trajectories of representative droplets over 100s performed for each chitosan concentration. (v) Schematic illustrations of the expected structure of particles at the oil-water interface and within the continuous aqueous phase depending on the chitosan concentrations used to prepare the emulsions. Emulsions were obtained from 8.7wtsusp% silica suspensions at pH 5.5. The oil-to-water ratio of the emulsion was 1:9.

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Figure 6: Stability of emulsions assessed through the evolution of the droplet size distributions over time. Graphs at the left and right show size distributions in the form of discrete and cumulative volumes, respectively. The oil-in-water emulsions were prepared with 8.7wtsusp% silica suspensions modified with different amounts of chitosan at a pH of about 5.5: (a) 0.2wt%, pH 5.49; (b) 1wt%, pH 5.48; (c) 5wt%, pH 5,51; (d) 10wt%, pH 5.34. The oil-to-water ratio was 1:9.

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Figure 7: (a) Storage G’ and loss G” moduli of oil-in-water emulsions prepared from 8.7wtsusp% silica suspensions modified with 1wt% or 5wt% of chitosan at pH 5.5. Measurements were conducted directly after emulsification. (b) Storage Gi’ and loss Gi” moduli of the oil-water interface formed using corn oil as upper phase and 1wt% silica suspensions modified with 1wt% or 5wt% of chitosan at pH 5.5 as bottom phase. (c) Schematics of the main stabilization mechanisms proposed for emulsions containing different chitosan concentrations: Pickering stabilization for 1wt% of chitosan and network formation for 5wt% of chitosan.

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Figure 8: Cryo-SEM image of the percolating network of particles responsible for the stabilization of oil droplets in emulsions containing 5wt% of chitosan. The oil-in-water emulsion was prepared from 8.7wtsusp% silica suspension at a pH of 5.5. The oil-to-water ratio of the emulsion was 1:9.

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Table of Content Figure 85x47mm (300 x 300 DPI)


Pickering and Network Stabilization of Biocompatible Emulsions Using

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