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

Abstract

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.

Liquid–liquid phase separation-inspired design of biomaterials

“…The low interfacial tension between two immiscible aqueous phases often results in the dynamic interfacial adsorption and detachment of colloidal nanoparticles. 24 This low energy barrier for colloidal detachment fails to effectively prevent the coalescence of the water/water (W/W) droplets. To overcome this limitation, I propose the use of protein nanofibrils as stabilizers for W/W droplets.…”

“…As evidence, we successfully developed mini-scaffolds of fibrillosomes for long-term culture of tumor spheroids (SGC-996) lasting over 30 days. 24 The high permeability of oxygen and nutrients across fibrillosomes supports the growth of large tumor spheroids, with reduced hindrance to cell fusion compared to hydrogel particles. These unique features of fibrillosomes make them promising candidates for more efficient applications in anticancer drug screening and tissue engineering.…”