In vivo behavior of systemically administered encapsulin protein nanocages and implications for their use in targeted drug delivery

Encapsulins, self-assembling protein nanocages derived from prokaryotes, are promising nanoparticle-based drug delivery systems (NDDS). However, the in vivo behavior and fate of encapsulins are poorly understood. In this study, the interactions between the model encapsulin from Thermotoga maritima (TmEnc) and key biological barriers encountered by NDDS are probed. Here, a purified TmEnc formulation that exhibits colloidal stability, storability, and blood compatibility is intravenously injected into BALB/c mice. TmEnc has an excellent nanosafety profile, with no abnormal weight loss or gross pathology observed, and only temporary alterations in toxicity biomarkers are detected. Notably, TmEnc demonstrates immunogenic properties, inducing the generation of nanocage-specific IgM and IgG antibodies, but without any prolonged pro-inflammatory effects. An absence of antibody cross-reactivity also suggests immune-orthogonality among encapsulin systems. Moreover, TmEnc forms a serum-derived protein corona on its surface which changes dynamically and appears to play a role in immune recognition. TmEnc's biodistribution profile further reveals its sequestration from the blood circulation by the liver and then biodegrades within Kupffer cells, thus indicating clearance via the mononuclear phagocyte system. Collectively, these findings provide critical insights into how encapsulins behave in vivo, thereby informing their future design, modification, and application in targeted drug delivery.