Bioluminescent Trace Labeling of Porous Protein Microcrystals for Evaluating Their Pharmacodynamics

In this exploratory pilot study, we evaluated bioluminescent trace labeling as a strategy for monitoring the in vivo distribution of cross-linked porous protein microcrystals grown from the CJ protein. CJ crystals doped with CJ-SpyTag003 were functionalized with SpyCatcher003-NanoLuciferase and imaged after intravenous administration in BALB/c mice. In vitro experiments confirmed that SpyCatcher-fusion proteins could be retained by CJ-SpyTag003 crystals and that NanoLuciferase remained active after association with the crystal scaffold. Following tail-vein injection, whole-animal IVIS imaging revealed early thoracic signal consistent with first-pass pulmonary exposure, followed by rapid signal decay. A subset of animals showed persistent hindlimb-associated signal, but terminal ex vivo imaging at 72 h showed no statistically significant organ-level differences after sensitivity analysis excluding one extreme muscle-associated value. These results demonstrate the feasibility of NanoLuciferase-based trace labeling for longitudinal imaging of CJ microcrystals while highlighting important interpretive limitations of substrate-dependent bioluminescence, including furimazine delivery, local substrate pooling, reporter stability, and the inability of bioluminescence alone to distinguish intact crystals from released or surface-associated NanoLuciferase. Overall, this work provides an initial in vivo imaging framework for CJ protein microcrystals and identifies particle size, reporter stability, and orthogonal tracking as priorities for future pharmacokinetic studies.