Loading Of Biomolecules Inside The Cavity Of Polyelectrolyte Capsules

One important feature of capsules lies in the possibility to load desired cargo molecules into their cavity. Encapsulation allows for protection, both of the cargo materials from inactivation and dilution effects inside living cells, and the cell itself from the possible toxic effect of the cargo. In recent years, numerous approaches have been envisaged to entrap different types of materials, such as small crystals, fluorescent dyes, drugs, and biomolecules (e.g. DNA, small-interfering RNA (si-RNA) and enzymes) within capsules.

One strategy employs the charged properties of enzyme crystals and of cells^[ to use them as templates onto which oppositely charged polyelectrolyte pairs can be directly adsorbed. Remarkably, the functional properties of the enzymes trapped in the capsule cavities are preserved during the LbL assembly process. The activity of coated enzymes is fully retained after exposure to proteases, whereas uncoated enzymes are deactivated by more than 90% in a short period of time, which demonstrates that the cargo inside the capsule cavity is protected from enzymatic degradation. Also, cells embedded in polyelectrolyte walls are able to maintain their viability, functionality, and normal exchange of nutrients and waste. However, one limit of the method lies in the impossibility of synthesizing monodisperse particles with regular shapes since the final embedded product strongly depends on the template.

Another method exploits the possibility to load materials into the assembled polyelectrolyte capsules immediately after the dissolution of the core (post-loading method). In particular, the wall permeability of capsules can be reversibly switched from the opened to the closed state through variation of pH value or through variation of the solvent polarity (fig 4a). The capsules can be loaded at low pH or in the presence of ethanol (open state), as in this state the polyelectrolyte network of which their walls are composed is swollen and thus permeable, so that molecules can diffuse in. After increasing the pH value or dispersing the suspension in the original medium (without ethanol), the polyelectrolyte network shrinks, the walls are no longer permeable, and thus the loaded materials are retained inside the cavity (closed state). One of the main advantages of encapsulating molecules using the post-loading method relies on the suitability of commercially available monodisperse templates which allow for the synthesis of monodisperse polyelectrolyte capsules. Obviously, materials sensitive to pH changes are not suitable for loading under these conditions. However molecules that are too big to diffuse through the walls in closed state can be loaded.

Fig. 4. (a) Encapsulation (post-loading) – permeation and encapsulation of biomolecules into multilayered PE capsules. (Left) Capsules suspended in aqueous solution are impermeable to biomolecules (closed-state). (Center) Diffusion of biomolecules into the cavity after reducing the pH of the solution or by the addition of ethanol (EtOH) (open-state). (Right) Encapsulated biomolecules after resuspension in water medium (closed-state) and after washing away all biomolecules outside the capsule. (b) Encapsulation (coprecipitation) (i) Spherical CaCO₃ microparticles comprising the cargo molecules are fabricated by precipitation from supersaturated CaCl₂ and Na₂CO₃ solution in the presence of the cargo molecules. (ii–iv) Cyclic addition of oppositely charged polymer layers by electrostatic adsorption. (v) Core dissolution by EDTA treatment to obtain PE capsules with cargo molecules inside their cavity. The capsules are not drawn to scale.

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An alternative to the above mentioned procedures has recently been proposed. It is based on the coprecipitation of the cargo onto porous templates, such as carbonate crystals, followed by the multilayer assembly of polyelectrolyte pairs (pre-loading method) (fig 4b)Some limits of this method are the difficulty in obtaining colloidal crystals of low dispersity in the desired size range and in the unavailability of commercially synthesized carbonate templates. Nevertheless, these cores can be easily dissolved in mild conditions (e.g. by using EDTA or low pH) without affecting the encapsulated materials .