Reprogramming the flavo-encapsulin from Thermotoga maritima to a novel protein nanocage platform for potent photodynamic therapy.

Encapsulins are protein nanocages consisting of a single monomer protein and have gained increasing interest as a platform for diverse biological nanotools due to their ease of production and robust stability. We found that the Thermotoga maritima encapsulin (TmEnc) recombinantly expressed in Escherichia coli possessed intrinsically bound riboflavin, as confirmed by LC-MS analyses. By denaturing the native nanocage under acidic pH followed by dialysis at neutral pH, we generated the apo-TmEnc and subsequently reconstituted it with exogenously supplied riboflavin. To functionalize TmEnc nanocage for biomedical applications, we displayed the EGFR-targeting peptide AEYLR on its surface by genetically fusing the peptide sequence to either the C-terminus or an exposed loop region of TmEnc. We further reprogrammed TmEnc as a photodynamic therapy (PDT) nanocarrier by loading it with the photosensitizer rose bengal during the reconstitution process. The rose bengal-loaded, AEYLR-modified TmEnc exhibited marked photocytotoxicity toward EGFR-overexpressing cancer model cells. Given its high rose bengal loading efficiency and excellent reconstitution yield, the resulting flavin-depleted, photosensitizer-reconstituted TmEnc nanocage represents a promising and cost-effective foundational platform for PDT applications.