Investigating the utility of encapsulin protein nanocages as bio-inspired delivery systems

Encapsulin protein nanocages are a new class of pseudo-organelles found inside prokaryotes. They self-assemble from multiple protein subunits into hollow nanoparticle-like structures (18-42 nm) that exhibit robust stability and excellent solubility and can be readily modified via genetic and/or chemical strategies. Uniquely, encapsulins assemble around cargo proteins tagged with an encapsulation signal peptide (ESig), selectively packaging them. Based on these distinct features, encapsulins have been recently employed as bio-inspired delivery systems for anticancer drugs and vaccines. However, a complete understanding of their biological interactions and fate are still lacking. In this thesis, the behaviour of encapsulins (and engineered variants) is investigated in both in vitro and in vivo models of neuroblastoma - a paediatric cancer of the nervous system.

Encapsulin from the bacterium Thermotoga maritima (Tm) was genetically engineered to display cancer-targeting RGD peptides on its outer surface (RGD-Enc). Neuroblastoma cells were treated with fluorescently labelled Enc or RGD-Enc at varying concentrations for different time periods, and their cellular internalisation was visualised and quantified by confocal laser scanning microscopy and flow cytometry. RGD-Enc demonstrated significant targeted cellular uptake, whereas only negligible uptake was observed for unmodified Enc (wt-Enc). Nanocages were then systemically administered into a tumour-bearing mouse model. Whole-body and whole-organ fluorescent imaging revealed that wt-Enc and RGD-Enc were both unable to accumulate within tumours and were primarily deposited within the liver, suggesting clearance by the mononuclear phagocytic system. Importantly, the treated mice displayed no behavioural changes or hypersensitivity reactions up to 24h post-injection, which confirmed safety. Together, this in vitro uptake and in vivo biodistribution studies further our understanding of encapsulins and lay the foundation for the future development of encapsulin-based delivery systems.

Neuroblastoma has elevated copper levels that drive its progression, and thus copper-sequestering drugs have been shown to suppress tumour activity (i.e., anti-copper therapy). Native Tm encapsulin packages the iron-storage proteins and allows it to sequester (via surface pores) and store toxic intracellular iron ions, protecting its prokaryotic host from oxidative stress. Inspired by this natural phenomenon, we sought to reprogram Tm encapsulin into Copper-Sequestering Protein Nanocompartments (CSPN) for potential use in copper-chelation and copper-ionophoric cancer therapy. Herein, ESig-tagged copper-storage proteins from the methane-oxidising bacterium Methylosinus trichosporium were loaded into encapsulins. The resulting CSPNs were able to sequester and store copper within its inner cavity when produced inside Escherichia coli that was cultivated in copper-supplemented media. Alternatively, the CSPN could be extracted from E. coli, purified and successfully demonstrated excellent functionality in sequestering exogenous copper.