Quantum dot-NanoLuc bioluminescence resonance energy transfer enables tumor imaging and lymph node mapping in vivo

Abstract

A small luciferase protein (Nluc) was conjugated to QDs as a bioluminescence resonance energy transfer (BRET) pair. The conjugate showed 76% BRET efficiency and lymph node mapping was successfully performed. The cRGD peptide was conjugated to QD-Nluc for tumor targeting. The self-illuminating QD-Nluc showed excellent energy transfer in a living system and offered an optimal tumor-to-background ratio (>85).

Fig. 1

Characterization of bioluminescent-QD conjugates based on BRET. (a) Synthesis of QD-Nluc and QD-Nluc-cRGD conjugates. (b) Absorption and emission spectra of QD705 (λ_ex = 465 nm), and bioluminescence of furimazine catalyzed by Nluc. (c) Bioluminescence emission spectrum of QD-Nluc in PBS. (d) Gel electrophoresis analysis of the conjugates; lane 1 is purified QD-Nluc conjugate; lane 2 is purified QD-Nluc-RGD conjugate, and lane 3 is unconjugated QD705.

Fig. 2

In vivo luminescence lymph node imaging. (a) Bioluminescence imaging of lymphatic basins in a mouse injected with QD-Nluc intradermally in a hind paw. (b) Fluorescence image of lymph nodes excised from the mouse injected with QD-Nluc in four paws (see Fig. S2); L, left; PO, popliteal lymph node; LU, lumbar lymph node; IL, iliac; AX, axillary lymph node; R, right. (c–d) Histological images of lymph node slices from a mouse in (a); (c) PO lymph node from injected mouse exhibited bright signal from QD705 (red) whereas no significant emission from QD705 was observed in PO lymph node from non-injected mouse (d). DAPI (blue) represents nuclease, scale bar: 20 μm.

Fig. 3

Fluorescence and luminescence imaging of U87MG cells with QD-Nluc-cRGD conjugate. (a–c) Confocal imaging; (a) cells were incubated with QD-Nluc-cRGD for 3 h, (b) cells were incubated with QD-Nluc for 3 h and (c) control cells without any conjugates, scale bar: 10 μm. (d) Luminescence images of labeled cells acquired without any filter (left) and with a filter (690–710 nm, right); cells were incubated with QD-Nluc (tube 1) and QD-Nluc-cRGD (tube 2). (e) Cell viability of U87MG cells evaluated by MTT assay in a dose-dependent manner of QD-Nluc and QD-Nluc-cRGD conjugates. Data represent mean ± s.d. (n = 4).

Fig. 4

In vivo fluorescence imaging of U87MG tumor-bearing mice. (a,b) Time-dependent fluorescence imaging of U87MG tumor-bearing mouse intravenously injected with (a) QD-Nluc-cRGD or (b) QD-Nluc at 5 min, 30 min, 1 h and 2 h p.i..

Fig. 5

Luminescence imaging of U87MG tumors in mice with QD-Nluc-cRGD. a,b) Time-dependent bioluminescence imaging of U87MG tumor-bearing mouse intravenously injected with QD-Nluc-cRGD (the same mouse as Fig. 4a) and single injection of furimazine substrate. Images in a) acquired without any emission filter with acquisition time: 1 s (5 min), 1 s (30 min), 1 s (1 h) and 10 s (2 h). Images in b) acquired with emission filter (690–710 nm) with acquisition time: 10 s (5 min), 10 s (30 min), 30 s (1 h) and 3 min (2 h).

Fig. 6

Ex vivo fluorescence imaging of organs and tumor tissues. a,b) Fluorescence images of organs excised from the mice in Fig. 4 injected with QD-Nluc (a) and QD-Nluc-cRGD (b). c,d) Histology of frozen U87MG tumors slices from a mouse injected with QD-Nluc (c) and QD-Nluc-cRGD (d). Scale bar: 10 μm.