Modular PEGylation Confers Reduced Immune-Cell Uptake and Enhanced Pharmacokinetic Performance to Encapsulin Protein Nanocages

Encapsulins are self-assembling prokaryotic protein nanocages with growing potential as systemic drug delivery systems, but their pharmacokinetic behaviour remains poorly characterised, and rapid immune recognition and clearance may limit delivery to target tissues. Here, we show that modular PEGylation of a SpyCatcher- decorated encapsulin Alkaliphilus metalliredigens (Am-S) markedly reduces macrophage uptake and extends systemic circulation. Site-directed surface PEGylation using SpyTagged PEG achieved ∼82% conjugation efficiency, corresponding to an estimated average of ∼49 PEG chains per 60-subunit Am-S nanocage, without compromising nanocage assembly, morphology, or colloidal stability. PEGylated Am-S also retained solubility and protein integrity following freeze–thaw cycling and six months of storage. When interacted with RAW 264.7 macrophages in vitro , PEGylation substantially reduced nanocage association and internalisation relative to non-PEGylated nanocages. Following intravenous administration in BALB/c mice, PEGylated nanocages exhibited markedly prolonged circulation, with >50% of the injected dose remaining after 1 h compared with 2.6% for non-PEGylated Am-S, and a circulatory half-life of 1 h 43 min. To our knowledge, this represents the first pharmacokinetic characterisation of an encapsulin nanocage. Together, these findings demonstrate that controlled PEGylation can substantially reduce macrophage interactions and prolong encapsulin circulation in vivo .