Bioluminescent trace labeling of porous protein microcrystals for evaluating pharmacodynamics

ABSTRACT: In the present study, we investigated the biodistribution and persistence of cross-linked porous protein microcrystals, grown from CJ protein, in vivo using bioluminescence imaging. By coupling the NanoLuc luciferase enzyme to the CJ crystal scaffold, we sought to determine whether these protein-based crystalline materials remain systemically dispersed, accumulate in specific organs, or become locally retained following intravenous injection. Ex vivo imaging of dissected organs at 72 hours post-injection revealed relatively uniform radiance across most tissues; however, persistent signal in the hindlimbs of several animals suggested partial entrapment of crystals within skeletal muscle microvasculature. Statistical evaluation using ANOVA, Kruskal–Wallis, and post hoc testing confirmed that, while organ-level differences were not significant after outlier removal, the spatial pattern of emission observed in whole-animal imaging supports a model of microvascular retention. These results establish the foundation for considering CJ microcrystals as potential localized depot systems capable of sustained, site-specific release of therapeutic payloads. Beyond biodistribution, this work highlights the physical and physiological determinants that govern the fate of solid, protein-based micro- and nanoparticles following systemic administration.