Bioluminescence imaging (BLI) provides various advantages over fluorescence imaging. Since BLI does not require excitation light, the technique does not suffer from photobleaching and autofluorescence, and therefore better signal-to-noise ratios can be achieved in many heterogeneous environments. Recent advances in luciferase engineering have further provided a suite of bright and stable reporters. However, the full potential of these luciferases for nanoparticle tracking has not been thoroughly evaluated. In this study, we aimed to examine the applicability of a protein nanoparticle platform (E2) decorated with two bioluminescent proteins, NanoLuc (Nluc) and Akaluc, for in vitro and in vivo imaging. The bioluminescent proteins were engineered to attach to E2 (Nluc-E2/Akaluc-E2) using the SpyCatcher-SpyTag strategy, yielding a complex with a final diameter around 45 nm. Nluc-E2 and free Nluc demonstrated comparable luminescent signals at pH 7.4. Surprisingly, luminescence was greatly increased at low pH for Nluc immobilized on the nanoparticle, with Nluc-E2 showing two orders of magnitude higher radiance than free Nluc at pH 5.0. However, the large luminescence differences at pH 5.0 were not seen for Akaluc-E2. We also examined dendritic cell uptake of these particles and demonstrated that uptake of Nluc-E2 by DCs showed higher luminescence over time compared to uptake of free Nluc. In vitro and in vivo imaging were further performed with these bioluminescent sensors. Our results suggest that E2-immobilized Nluc is an attractive platform for monitoring DC internalization and other cellular imaging studies.

Immobilizing bioluminescent proteins on a nanoparticle platform for improving imaging

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.