Attoliter protein nanogels from droplet nanofluidics for intracellular delivery

Microscale hydrogels consisting of macromolecular networks in aqueous continuous phases have received increasing attention because of their potential use in tissue engineering, cell encapsulation and for the storage and release of cargo molecules. However, for applications targeting intracellular delivery, their micrometer-scale size is unsuitable for effective cellular uptake. Nanoscale analogs of such materials are thus required for this key area. Here, we describe a microfluidics/nanofluidics-based strategy for generating monodisperse nanosized water-in-oil emulsions with controllable sizes ranging from 2500 +/- 110 nm down to 51 +/- 6 nm. We demonstrate that these nanoemulsions can act as templates to form protein nanogels stabilized by supramolecular fibrils from three different proteins. We further show that these nanoparticles have the ability to penetrate mammalian cell membranes and deliver intracellular cargo. Due to their biocompatibility and lack of toxicity, natural protein-based nanoparticles present advantageous characteristics as vehicles for cargo molecules in the context of pharmaceutical and biomedical applications.