Water-in-water Pickering emulsion stabilized by polydopamine

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Water-in-water Pickering emulsion stabilized by polydopamine particles and crosslinking Jianrui Zhang, Jongkook Hwang, Markus Antonietti, and Bernhard V. K. J. Schmidt Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01301 • Publication Date (Web): 05 Nov 2018 Downloaded from http://pubs.acs.org on November 6, 2018

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Water-in-water Pickering emulsion stabilized by polydopamine particles and crosslinking Jianrui Zhang,† Jongkook Hwang,† Markus Antonietti,† Bernhard V. K. J. Schmidt†,* † Max-Planck

Institute of Colloids and Interfaces; Department of Colloid Chemistry, Am

Mühlenberg 1, 14476 Potsdam, Germany

ABSTRACT

All aqueous multi-phase systems have attracted significant attention recently, in particular water-in-water Pickering emulsions. In here, polydopamine nanoparticles (PDP) are investigated as stabilizer for dextran and poly(ethylene glycol) (PEG)-based aqueous emulsions. Remarkably, stable emulsions are obtained from the all-biocompatible materials that can be broken either via dilution or surfactant addition. Further crosslinking of PDP via poly(acrylic acid) and carbodiimide strengthens the stability of emulsion droplets in a colloidosomes-like structure. After crosslinking, demulsification via dilution or surfactant addition was largely hindered. The PDP-mediated formation of all aqueous emulsions is expected to be generalized to different types of water-in-water emulsions with other polymers and offer new opportunities in surface modification and microencapsulation.

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Introduction Water-based polymer systems are not only omnipresent in biology, but also in synthetic polymer science and many consumer products rely to a large extent on the utilization of biomacromolecules in water. Recent highlights are illustrating the shift of interest towards this segment of polymer science, including the synthesis of stimuli-responsive hydrophilic polymers and materials,1-3 smart hydrogels4, 5 or block copolymer self-assembly in water6, 7. Moreover, applications in the biomedical field such as drug-delivery,8,

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nano reactors10 or

tissue-engineering11 have recently driven the research towards water-based polymer systems. A water-based polymer system of particular interest and unexpected potential is the aqueous two-phase system (ATPS) that can be prepared via mixing of two aqueous solutions of watersoluble polymers, which turn biphasic when exceeding a critical polymer concentration.12-14 ATPS are utilized commonly in the literature for the partitioning and purification of enzymes as well as other biological vectors, for other separation tasks or in biotechnology.15-20 Tightly related to ATPS are water-in-water (w/w) emulsions that found significant attention recently due to their potential applications in cosmetics or food, possibly as an alternative to deliver ingredients with preferred solubility in the dispersed phase. Several examples of ATPS-based emulsions have been described in the literature. The dextran/poly(ethylene glycol) (PEG) system is a model case and has been studied frequently for the formation of all aqueous emulsions: In an earlier investigation of w/w emulsions formed by mixing aqueous solutions of dextran and PEG, it was shown that latex particles adsorbed spontaneously at the interface.21 Moreover, ultrathin plate-like colloidal particles,22 polysaccharides23 and cellulose nanocrystals24 can be used to stabilize the dextran/PEG emulsion system as well. An example of particular interest is the spontaneous adsorption of protein microgels at the w/w interface that led to very efficiently stabilized water compartments.12, 25 Notably, also other polymer mixtures were investigated, e.g. xyloglucan/amylopectin26 as well as the study about phase separation behavior of different components in ATPS by Whitesides and coworkers.27 2 ACS Paragon Plus Environment

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For ATPS, the interface between the two phases is rather undefined and spans a region larger than the characteristic correlation length of the polymer solutions. Thus, in comparison to oil-water emulsions, w/w emulsions cannot be stabilized by surfactants, as they miss the scale of the phase boundary.21,

23, 25, 28-31

On the contrary, solid particles adsorbed at the

interfaces can be exceptionally efficient stabilizers forming so-called Pickering emulsions,32, 33

and nanoparticles can bridge the correlation length of polymer solutions and are thereby the

best option for w/w emulsions. The presence of the solid particles at the interface inhibits coalescence of droplets, leading to efficient stabilization. Even though the interfacial tension between two aqueous polymer solutions is orders of magnitude smaller than between oil and water,34, 35 their binding energy is orders of magnitude larger than the thermal energy. Hence particles located between two aqueous phases are incapable of leaving spontaneously. It was shown that submicrometer solid particles adsorb irreversibly to the interface of the two aqueous phases opening up the possibility to create stable water-in-water emulsions without gelling one of the phases.23,

25, 34

Contrary, another option to stabilize water-in-water

emulsions is by gelling one or both of the phases. Recently, poly(dopamine) (PD) has tuned into an interesting choice for polymer and materials chemistry.36, 37 For example, it was employed in the formation of capsules38, 39 or as coating on various surfaces.38, 40, 41 In such a way, Caruso and coworkers formed PD capsules for drug-delivery42 that could be used to transport and release Doxorubicin via intracellular pH trigger. One remarkable feature of PD is its biocompatibility43 and its adhesion to almost every surface because of a combination of high hydrophobicity and polycationic character. Nevertheless, one has to keep in mind that the molecular structure of PD is rather illdefined.44,

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In the present study, such biocompatible poly(dopamine) particles (PDP) are

applied to stabilize water-in-water emulsions, where PDP acts as solid stabilizer adsorbed at the interface. As a result, control of shell characteristics requires understanding of the process



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of colloidal assembly at interfaces. In addition the system was further developed towards colloidosome formation via crosslinking. Colloidosomes, i.e. microcapsules made up by a shell of colloidal particles are well studied potential carriers of active compounds for various applications as such. Commonly, colloidal shell synthesis can be conducted via the self-assembly of appropriate nanoparticles at oilwater interfaces and subsequent crosslinking.46-49 Specifically, the colloidosome shell is composed of impermeable regions (the particles) and permeable ones (the interstitial volume). In many cases, they are stabilized through fusion,50 while in others they are adsorbed irreversibly onto a polymer gel scaffold.51-53 Recently, Mann and coworkers utilized ATPS to form colloidosomes from poly(styrene) latex particles.54

Scheme 1. Overview of the dextran/PEG water-in-water emulsion formation employing poly(dopamine) particles (PDP) and crosslinking with poly(acrylic acid) (PAA) / 1-ethyl- 3-


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(3-dimethylaminopropyl)carbodiimide (EDC). Insets: Structures of dextran (idealized), PEG, PD (idealized), PAA and EDC.

Herein, the formation of all aqueous dextran−PEG emulsions in the presence of PDP is investigated employing biocompatible components only. Studies of the formed emulsions are performed via confocal laser scanning microscopy (CLSM), optical microscopy (OM), cryoscanning electron microscopy (cryo-SEM) and tensiometry. The stability of the formed emulsions is studied with respect to pH, dilution and addition of surfactants. To inhibit demulsification of the Pickering emulsion, crosslinking of the solid PDP by using poly(acrylic acid) (PAA) and water-soluble 1-ethyl- 3-(3-dimethylaminopropyl)carbodiimide (EDC) is performed to lock-in the surface structure of emulsion droplets. After crosslinking, the stability of the colloidosome-like structure is probed again. Such types of aqueous emulsions and colloidosomes could potentially provide new opportunities for applications, e.g. in cosmetics or food products, possibly as an alternative to deliver ingredients with preferred solubility in the dispersed aqueous phase.

Experimental Part Materials Cetyltrimethyl ammonium bromide (CTAB; analytical grade, Fluka), dimethylsulfoxide (DMSO; Acros, extra dry, 99%), dextran (40k, analytical grade; 100k, analytical grade, all from Sigma Aldrich), dopamine (98%, Sigma Aldrich), ethylenediamine resin (polymerbound, 4.0-5.7 mmol/g, Sigma Aldrich), 1-ethyl-3-(3-dimethylaminopropyl carbodiimide hydrochloride (EDC; >98%, Sigma Aldrich), fluorescein isothiocyanate (FITC; 90%, Sigma Aldrich), hydrochloric acid (HCl; fuming, Carl Roth), poly(acrylic acid) (PAA; 450k, 5 ACS Paragon Plus Environment


analytical grade; 1250k, analytical grade, all from Sigma Aldrich), poly(ethylene glycol) (PEG; 20k, analytical grade; 35k, analytical grade, all from Sigma Aldrich), poly(ethylene glycol) diamine (NH2-PEG-NH2; 20k, analytical grade, Sigma Aldrich), sodium hydroxide (NaOH; 98%, Sigma-Aldrich) and sodium dodecyl sulfate (SDS; analytical grade , Fluka) were used without further purification. Milli-Q water was obtained from an Integra UV plus pure water system by SG Water (Germany).

Preparation of polydopamine particles (PDP).55 In a typical synthesis of PDP, aqueous ammonia solution (NH4OH, 0.75 mL, 28-30%) was mixed with ethanol (40 mL) and deionized water (90 mL) under mild stirring at room temperature for 30 min. Dopamine hydrochloride (0.5 g) was dissolved in deionized water (10 mL) and then injected into the mixture. The color of this solution immediately turned to pale brown and gradually changed to dark brown. The reaction was allowed to proceed for 30 h under air. The PDP was obtained by centrifugation and washed with water for three times. The fabricated PDP was dried in the oven at 80 °C for 24 h (yield: 0.38 g PDP). For the use as Pickering stabilizer a stock of PDP suspension was prepared applying ultrasound (Elmasonic S30H).

FITC-labeled PEG. In a dry, argon purged 25 mL round bottom Schlenk flask, NH2-PEG-NH2 (20k, 0.5 g, 0.025 mmol, 1 eq.) was dissolved in dry DMSO (6 mL). At first, FITC (9.735 mg, 0.05 mol, 2 eq.) was dissolved in dry DMSO (1.0 mL) in the dark and then added to the reaction mixture. The reaction mixture was stirred at ambient temperature for 24 hours. Ethylenediamine resin ( polymer-bound, 100 mg) was added and the reaction mixture was stirred for an additional 24 6 ACS Paragon Plus Environment

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h. The reaction mixture was filtered off and the solution was dialyzed against deionized water for three days followed by lyophilization to afford FITC labeled PEG (0.189 g, 10.2 µmol, 38% recovery, Mn = 18 400 g mol−1, PEG standard in THF, Đ = 1.3) as an orange powder.

Exemplary Preparation of Water-in-water Emulsions. Solutions of dextran and PEG were prepared by dissolving the powder in Milli-Q water at neutral pH with stirring. Concentrations of PEG (CPEG) and dextran (CDex) are indicated as weight percentages. An example emulsion was formed by mixing PEG35k (CPEG=7 wt%, 0.90 g) and dextran40k (CDex=3 wt%, 0.40 g) in PDP suspension (0.2 g/L, 3 mL). After mixing, the mixtures were shaken by hand. In this way, a stable dextran-in-PEG emulsion with broad distribution of droplet sizes could be achieved. In order to decrease dispersity of droplet sizes, the procedure was evaluated further. Vortex treatment for 30 s led to emulsion with improved dispersity in droplet size. Other emulsions were formed according to Table S1.

Preparation of Crosslinked Water-in-water Emulsions. A stock solution of PAA450k (100 mM) and freshly-prepared EDC (120 mM) was prepared in Milli-Q water. To obtain crosslinked PDP in water phases, 0.5 mL of PAA and EDC solution were added into as-formed emulsions stabilized by PDP. First, PEG35k (CPEG=7 wt%, 0.90 g) / dextran40k (CDex=3 wt%, 0.40 g) emulsions were prepared and then PAA450k solution (0.5 mL) and several droplets of fresh EDC solution (0.4 mL) were added into stable PEG/dextran emulsions, crosslinked PDP surrounding emulsion droplets were fabricated via vortex for 15s.

Demulsification of Various Types of Emulsions. 7 ACS Paragon Plus Environment


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Here, PEG (CPEG=7 wt%, 0.90 g) / dextran (CDex=3 wt%, 0.40 g) water-in-water solutions were stabilized by PDP as mentioned above. A droplet of the emulsion (~0.2 mL) was placed on a microscopy slide and Milli-Q water (~0.2 mL) was added to dilute the emulsion. In the case of surfactants, SDS or CTAB (4 mM, 0.3 mL) were used for 3 mL of non-crosslinked emulsions as well by directly mixing with original emulsions.

Characterization Methods The interfacial tensions between polymer solutions and water were determined by the pendant drop method through droplet shape profile analysis (OCA instrument, Dataphysics ES, Germany) (Figure S7). First, the water solution with lower density, dextran solution or PEG solution was poured into a cuvette and a volume of ca. 20 µL aqueous solution containing PDP, and a distinct concentration of the other solution was injected into it by a syringe. Then, the droplet shape profile was analyzed to acquire the value of interfacial tension. At least three independent measurements were performed. To measure the threephase contact angle of PDP at the air−water interface, a silicon wafer was immersed into PDP suspension and left to equilibrate for 24 h. After equilibration, the wafer was washed by distilled water to remove excess particles and dried prior to use. The wafer was placed at the bottom of the stage and ca. 2 µL water droplet was placed gently on the wafer. The threephase contact angle was recorded using the same OCA instrument.

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The contact angle was

obtained by measuring three different spots on a wafer. The size and zeta potential of PDP under different pH values were measured by using a Zeta Nanosizer instrument (Malvern Instruments, UK), at a fixed scattering angle of 90°. All measurements were repeated at least three times. For the size of PDP the volume weighted particle size distribution was employed. Fourier transform infrared (FTIR) spectrometer (Tensor 27, Bruker, Germany) was used to characterize the PDP synthesized before and after 8 ACS Paragon Plus Environment

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crosslinking. TEM (JEM-2100, JEOL, Japan) and SEM (JSM-7500F) were used to visualize the morphology of PDP before and after crosslinking. Cryo-SEM technique was used to visualize the emulsion droplet surface with a cryo chamber from Gatan (ALTO 2500). The prepared fresh dispersion was applied to a copper sample holder, and then the sample holder was put into chamber. Finally, the sample was fractured and imaged. Fluorescent images were obtained by a confocal laser scanning microscope (CLSM, TCS SP5, Leica, Germany). Prior to visualization, FITC-labeled PEG (

Water-in-water Pickering emulsion stabilized by polydopamine

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