Bioluminescence resonance energy transfer- based NanoLuc-QDs for lymph node mapping and tumor imaging

Objectives: Near-infrared (NIR) fluorescence imaging (FLI) although sensitive is hampered by limited tissue penetration. While bioluminescence imaging (BLI) provides an internally activatable alternative, most BL proteins are limited by weak signal intensities and short-lived emission. Although, bioluminescence resonance energy transfer (BRET)-based NIR imaging can overcome these challenges, it has been limited by poor sensitivity and suboptimal photoproduction. In this study, newly discovered luciferase enzyme, known as NanoLuc (NLuc), with 150-fold brighter luminescence than traditional luciferases, was conjugated to QDs for enhanced in situ activatable NIR imaging.

Methods: NLuc (Em: 460 nm upon addition of substrate furimazine) was conjugated to polymer-coated CdSe/ZnS core-shell QD705 (Em: 705 nm) through carbodiimide-mediated amide coupling to form QD-NLuc. The system was characterized by gel electrophoresis, dynamic light scattering, and optical spectroscopy. For sentinel lymph node (SNL) mapping, QD-NLuc was intradermally injected into the hind paw before furimazine was intravenously injected.Moreover, cyclic arginine-glycine-aspartic acid peptide (cRGD) was conjugated to QD-NLuc for integrin α_vβ₃ targeted imaging in U87MG human glioblastoma xenografted mice. Both FLI (Ex: 450-480 nm, Em: 690-710 nm) and BLI (Ex: blocked, Em: Open; or Ex: Blocked , Em: 690-710 nm) were employed to monitor the accumulation of NLuc-QD-cRGD in the tumors at different timepoints post-injection (pi).

Results: QD-Nluc (21.2 ± 2.5 nm) displayed strong BL signal (due to NLuc peaking at 460 nm) upon addition of furimazine, alongwith a strong peak at 705 nm indicating highly efficient BRET (BRET ratio ~13.3, corresponding to 76 % efficiency). QD-NLuc nanoconjugates were stable in serum and phosphate buffer saline (PBS) after storage at 4 °C for 30 days. In vivo SNL mapping indicated strong BL and BRET signals in the popliteal (PO) lymph node, 5 min pi, devoid of any background signal. Sufficient signal was observed in vivo, >2 h after a single substrate injection, allowing for visualization of two more lymph nodes (PO on the opposite side and iliac). In additon, intravenously administered QD-Nluc-cRGD also displayed rapid and persistent uptake in U87MG tumors. Serial FLI indicated tumor-to-background ratios (TBRs) of 2.1 ± 0.1 (1 h) and 1.7 ± 0.3 (2 h), where the background refers to region surrounding the tumor. Weak fluorescence signal was observed in the tumors injected with QD-Nluc which can be attributed to enhanced permeability and retention (EPR) effect (TBRs ~ 1.4 ± 0.2 (1 h) and 1.0 ± 0.1 (2 h), n = 3). Enhanced BRET signals were also observed in the same mice, with TBRs ~ 102.3 ± 9.1 (5 min), 74.6 ± 1.8 (30 min), 61.3 ± 1.0 (1 h), and 23.0 ± 3.1 (2 h), under open emission filter. Intense luminescence signal was still observed after applying an emission filter, validating the BRET efficiency of the nanoconjugates in vivo (TBRs calculated to be 90.7 ± 6.3, 37.8 ± 2.7, 30.9 ± 5.2, and 10.6 ± 1.0 at 5, 30, 60 and 120 min pi, respectively). Passively targeted QD-Nluc could be better visualized via BLI; however, the TBRs were significantly lower than that obtained from active targeting group. Ex vivo imaging corroborated the in vivo results.

Conclusion: Herein, we report the first proof-of concept study, demonstrating the successful synthesis of QD-Nluc nanoconjugates for in vivo SNL mapping and tumor-targeted imaging. Bioluminescence and BRET-based imaging with these conjugates offered enhanced brightness and higher luminescence output in comparison to FLI alone. BLI of tumor-bearing mice injected with QD-Nluc-cRGD provided exceptionally high TBRs >85, with and without emission filter. Moreover, BRET signal remained high and slowly decayed, closely resembling the half-life of Nluc in vivo, indicating high integrity of the nanoconjugates under physiological conditions. Research Support: NIH, American Chemical Society and University of Wisconsin - Madison

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