Bioengineering Prokaryotic Nanocompartments into Photosensitizing Nanoparticles

Photodynamic therapy (PDT) is a selective and non-invasive cancer treatment. To destroy tumour cells, PDT relies on photosensitizers that are activated by light to convert the oxygen within tumour cells into toxic reactive oxygen species (ROS) that induce cell death. The fluorescent proteins KillerRed (KR) and mini-Singlet Oxygen Generator (mSOG) are unique biological photosensitizers that produce ROS when irradiated with light. Herein, encapsulins (ENC), a class of protein-based nanoparticles found in prokaryotes, were engineered to encapsulate KR or mSOG variants. All photosensitizer-loaded ENCs were produced in Escherichia coli and were ~30 nm in size, monodisperse and fluorescent. KR (Type I photosensitizer) generates ROS under green/yellow light. Upon activation with green light, KR-loaded ENC (KR-ENC) produced similar amounts of ROS as free KR, while unloaded ENC generated no ROS. We also visualised the rapid internalisation of KR-ENC by human brain cancer cells, confirming ENC’s feasibility as a nanocarrier for functional biological photosensitizers. mSOG (Type II photosensitizer) produces singlet oxygen (¹O₂) upon blue light irradiation. mSOG variants, mSOG-1 and mSOG-2, previously engineered for enhanced ¹O₂ generation were loaded into ENC. All mSOG-loaded ENCs produced ¹O₂ under blue light activation, with a mSOG-1-loaded ENC variant (mSOG-1-ENC) shown to be the most effective. Next, we evaluated the PDT killing effect of mSOG-1-ENC in a cell model of lung cancer. mSOG-1-ENC displayed no cytotoxicity in the dark, however, when activated with blue light, it caused a ~25% reduction in cancer cell viability. These results show light-activated mSOG-1-ENC’s photosensitizing capacity and its ability to mediate PDT.