All-aqueous Synthesis of Fibrillated Protein Microcapsules for Membrane-bounded Culture of Tumor Spheroids

Water-in-water (W/W) emulsion provides cytocompatible compartments for cell encapsulation and 3D printing of tissues. Formation of semi-permeable membrane at the W/W interface is critical to entrapment of cells in the droplet phase and supply of nutrients from the continuous phase. However, the adsorption of colloidal particles at the W/W interface is dynamic and reversible, which represents an inherent limitation for fabrication of mechanically robust cell-laden microcapsules. Here we demonstrate the preparation of thin, inflatable and semi-permeable microcapsules by using clusters of protein fibrils as building blocks and control their assembly at W/W interface. These fibril clusters are prepared by cross-linking lysozyme fibrils with multi-arm polyethylene glycol (PEG) through click chemistry. Compared to linear-structured fibrils, fibril clusters can strongly adsorb at W/W interface, and they packed into an interconnected meshwork to stabilize W/W emulsion. Moreover, when fibril clusters are complexed with calcium alginate, the hybrid microcapsules can bear a high osmotic gradient of 60 mOsm/Kg induced by dextran (Mw=500,000), and their surface area expand by over 100% without rupture. This all-aqueous biomaterial synthesis approach allows fabrication of mechanically robust capsules for long-time culture of SGC-996 tumor spheroids, with great potentials to be used in anti-tumor drug-screening and tissue transplantation.