ProLiCell was proposing to investigate the design of self-assembled protein nanosheets at liquid-liquid interfaces, resulting in interfaces displaying strong mechanical properties able to resist cell-mediated contractile forces. Understanding how the molecular structure of constituents of nanosheets impact on the mechanics of the assemblies allowed us to refine the design of corresponding interfaces whilst conferring tailored bioactive properties (including cell adhesion and cell specific ligands).
In the first part of this project, we developed methodologies allowing us to examine how mechanical properties of nanosheets assembled at liquid-liquid interfaces are regulated by molecular composition. To this aim, we set up a very sensitive interfacial rheology rig allowing the exchange of mobile phases and temperature modulation. We developed an analytical model allowing to quantitatively extract interfacial moduli from force probe microscopy experiments. In addition, we have set up a Langmuir-Blodgett trough system for liquid-liquid interfaces and optimised deposition conditions allowing the transfer of nanosheet for higher resolution imaging. Finally, we developed cell-based assays to characterise the impact of nanosheet composition and interfacial mechanics on cell adhesion and stem cell phenotype.
Having established these methodologies, the ProLiCell team has investigated how the chemistry of proteins and co-surfactants used for the assembly of nanosheets impact on their interfacial rheological properties, and in turn regulates the proliferation of adherent cells, including primary keratinocytes, mesenchymal stem cells, the epidermal HaCaT cell line, dermal fibroblasts and an HEK293 cell line often used for the production of biotherapeutics. Specifically, we investigated how supercharging globular proteins such as albumin impacts on their interfacial viscoelasticity and how further assembly with co-surfactants modulate such behaviour. In addition, we established that covalent co-surfactants underpin physical crosslinking of nanosheets via Van der Waals interactions and pi-stacking, conferring sufficient elasticity to the resulting interfaces. In addition, we showed how modulating the molecular weight of the proteins assembled alters interfacial toughness, presumably by providing soft domains that are able to dissipate energy upon local fracture of hard sub-domains. This results in assemblies sufficiently strong to resist cell-mediated forces.
In the final part of the project, ProLiCell developed a novel concept for the engineering of bioactive protein nanosheets and associated bioemulsions, using recombinant proteins that display tensioactive properties and bioactivity. This not only enables the simplified formation and biofunctionalisation of microdroplets, but also enable to bring sensitive bioactive elements to the surface of microdroplets in a scalable format. Using this approach, we demonstrated that bioactive microdroplet technologies (or bioemulsions) can capture important ligands to stimulate stem cells or capture them for separation. We have demonstrated the application of this technology to integrate in iPSC-derived platforms, including cerebral organoids.
