# Water-in-Water Emulsions as Templates for Microcapsules

Cells are complex structures with semipermeable membranes that enclose the cell contents and protect cells from the external environment while at the same time allowing selective transport of molecules into and out of the cell. In an attempt to mimic protocells, researchers have developed synthetic routes to generate microcapsules with membrane properties approximating the cell membrane. A common method to fabricate microcapsules is based on emulsion templates. Traditionally emulsions are formed by mixing of chemically dissimilar fluids such as water and oil in the presence of a stabilizer such as surfactants, particles, lipids, and block copolymers. However, for health foods, solvent-free cosmetics and applications that involve the use of chemically sensitive biomolecules such as proteins the use of an oil phase is undesirable. Replacing the oil phase with an aqueous phase to form emulsions templates can provide an attractive alternative for the above applications.

When mixing two or more aqueous solutions containing hydrophilic incompatible polymers, above a threshold polymer concentration, the solution will phase separate to form two distinct thermodynamic phases. If the phase separation occurs in the presence of a stabilizer, typically particles, stable water-in-water (W/W) emulsion will form. To arrest phase separation and form stable W/W emulsions, it is necessary that the particles adsorb to the W/W interface. Based on thermodynamic derivation, the change of the free energy ($\Delta G$) of the system due to adsorption of spherical particles with radius $r$ and contact angle $\theta$ can be calculated by

$\Delta G= \pi r^{2}\gamma_{w/w}(1-|cos \theta|)^{2}$  (1)

where $\gamma_{w/w}$ is the interfacial tension between two immiscible aqueous phases. To have particles irreversibly adsorb to the W/W interface (negative ?G), the interfacial tension should be greater than a threshold value set by thermal motion energy of particles; therefore larger particles are preferred (2). However, packing of larger particles at the surface of emulsion droplets typically result in low coverage, making it challenging to fabricate stable emulsions.

Song and coworkers developed a method to generate W/W emulsion templates that combines the advantages of large particles while allowing for effective packing at the W/W interface by starting with a monomers that grow to large mature fibrils. Their method utilized the assembly of pre-seeded protein fibrils at the surface of an emulsion droplet followed by conversion of additional protein monomers into anisotropic fibrils. The rationale behind their approach is that high aspect ratio fibrils will pack more efficiently at the emulsion interface in comparison to spherical particles. W/W dextran-in-poly ethylene oxide emulsions were generated by mixing aqueous solutions of polyethylene oxide and dextran, stabilized in the presence of protein fibrils. Conversion of the proteins monomer into fibrils was achieved by heating the emulsions mixture at 60 °C for three days.

A study of the emulsion stability as a function of the fibril growth stage revealed that only fully mature fibrils resulted in stable W/W emulsions. This confirmed the important role of fibrils in emulsion stabilization. Using microscopy imaging it was determined that majority of the fibrils were located at the emulsion interface thereby allowing high surface coverage of the fibrils at the emulsion interface.

For every aqueous two-phase system, there is a minimum interfacial tension ($\gamma_{w/w_{min}}$) below which the emulsions are often not stable even after adsorption of chemically-inert particles. Remarkably, the authors demonstrated that growth of bioactive protein fibrils at the emulsion interface is an alternative strategy to stabilize w/w emulsions, with interfacial tension below $\gamma_{w/w_{min}}$. Formation of a 2D colloidal network by crosslinking of the fibrils provides the additional energy needed to stabilize the emulsions below $\gamma_{w/w_{min}}$. Moreover, the emulsions can be converted into highly robust microcapsules by covalently crosslinking the 2D fibrils network. Lastly, the permeability of the microcapsule membrane was characterized and shown to be selective to molecules based on size.

Overall, the study presented here provides an attractive approach for capsule fabrication based on W/W emulsions templates. The use of self-growing protein fibrils as a stabilizer allow efficient packing at the droplet interface and results in higher emulsion stability compared to protein monomer stabilizers. In addition, the formation of multilayer fibrils network at the emulsion interface allow generation of stable W/W stable emulsion even at ultra-low interfacial tensions. Considering the mild preparation conditions of W/W emulsions stabilized by fibrils, we expect this system to have wide use in biomedical applications which require encapsulation and selective release of bioactive molecules.

(1) $\Delta G$ is the change in free energy of the system. $\Delta G$  tells us weather a process will be spontaneous or not; meaning will it simply happen on its own. If delta G is negative the process is spontaneous.

(2) At very low interfacial tensions, such as in water/water systems (1 ?N/m to 1000 ?N/m), reduction in interfacial tension contribution to $\Delta G$ term diminishes.