Self-luminescing BRET-FRET near-infrared dots for in vivo lymph-node mapping and tumour imaging

Abstract

Strong autofluorescence from living tissues, and the scattering and absorption of short-wavelength light in living tissues, significantly reduce sensitivity of in vivo fluorescence imaging. These issues can be tackled by using imaging probes that emit in the near-infrared wavelength range. Here we describe self-luminescing near-infrared-emitting nanoparticles employing an energy transfer relay that integrates bioluminescence resonance energy transfer and fluorescence resonance energy transfer, enabling in vivo near-infrared imaging without external light excitation. Nanoparticles were 30-40 nm in diameter, contained no toxic metals, exhibited long circulation time and high serum stability, and produced strong near-infrared emission. Using these nanoparticles, we successfully imaged lymphatic networks and vasculature of xenografted tumours in living mice. The self-luminescing feature provided excellent tumour-to-background ratio (>100) for imaging very small tumours (2-3 mm in diameter). Our results demonstrate that these new nanoparticles are well suited to in vivo imaging applications such as lymph-node mapping and cancer imaging.

Figure 1

Schematic of self-luminescing BRET-FRET near infrared (NIR) polymer nanoparticles. The biochemical energy generated from the Luc8-catalyzed oxidation of coelenterazine transfers initially to the MEH-PPV polymer and is then relayed to doped NIR775 dye to produce NIR emission. An amphiphilic polymer, PS-PEG-COOH, coats the nanoparticle to improve water solubility and biocompatibility. Tumor targeting ligands such as cyclic RGD peptides are conjugated to the nanoparticle surface for in vivo cancer imaging.

Figure 2

In vitro characterization of RET IR nanoparticles. (a) UV-Vis absorption and fluorescence emission spectra of RET₂IR NPs in PBS buffer. (b) Bioluminescence emission spectrum of RET₂IR NPs in PBS buffer. (c) Representative transmission electron microscopy (TEM) image of RET₂IR NPs. Scale bar = 200 nm. (d) Dynamic light scattering (DLS) measurement of four indicated NP formulations in water by lognormal size distribution. (e) Gel electrophoresis (0.5% agarose) analysis of RET NPs in tris-borate-EDTA (TBE) buffer: RET₁IR (lane 1), RET₁IR@cRGD (lane 2), RET₂IR (lane 3), RET₂IR@cRGD (lane 4). (f) Bioluminescent and NIR fluorescent intensity of RET₂IR NPs (1 μg) in mouse serum at 37°C from 0 h to 24 h. Data points represent mean±s.d. (n=3). (g) The NIR fluorescence signals of blood samples of mice injected with RET₁IR NPs (~20 μg) from 5 min to 24 h. Data represent mean±s.d. (n=4). (h) Viability values (%) of U87MG cells estimated by MTT assay versus incubation concentrations of RET₁IR NPs. Data represent mean+s.d. (n=3).

Figure 3

Fluorescence and bioluminescence imaging of lymph nodes in mice. (a) Fluorescence imaging of a mouse following sacrifice and necropsy 24 h after the tail-vein injection of RET₁IR NPs (~20 μg). Superficial skin was removed before imaging but peritoneum was left intact. Autofluorescence is coded in green and NPs signal in red; NL, neck lymph nodes; AX, axillary lymph node; LT, lateral thoracic lymph node; IN, inguinal lymph node; L, left; R, right. (b) Fluorescence image of lymph nodes excised from the mouse in (a): 1-4, NL; 5, AX (Left); 6, LT (Left); 7, LT (Right); 8, AX (Right); 9, IN (Left), 10 IN (Right). (c) Bioluminescence and (d) fluorescence imaging of lymphatic basins in a mouse 10 mins after the injection of RET₂IR NPs (~2 μg) intradermally in the forepaws. (e) Bioluminescence imaging of lymphatic basins in a mouse with injection of RET₂IR NPs (~2 μg) intradermally in the forepaws. All bioluminescence images were acquired with 10 s exposure time; PO, popliteal lymph node; LU, lumbar lymph node; L, left; R, right.

Figure 4

Fluorescence imaging of U87MG cells in vitro and in vivo with RET₁IR@cRGD NPs. (a-c) Live imaging of U87MG cells in vitro incubated with RET₁IR@cRGD NPs (4 μg) for (a) 2.5 h and (b) 24 h, or (c) incubated with RET₁IR NPs without cRGD for 24 h. Scale bar: 20 μm; excitation: 480/30 nm, dichroic beamsplitter: Q570LP, emission: D755/40M; objective: 20x; acquisition time: 1 s. (d, e) Time-dependent fluorescence imaging of U87MG tumor-bearing mouse (tumors are indicated by white arrows and circles, and are 4 mm in diameter) injected with (d) RET₁IR@cRGD or (e) RET₁IR NPs (each at ~50 μg) after 5 min, 2 h, 24 h and 48 h. Autofluorescence is coded in green and the unmixed polymer nanoparticle signal in red. (f) Fluorescence spectra of tissue autofluorescence (green) and the unmixed nanoparticle signal (red) in a living mouse. (g) ROI analysis of fluorescence intensity of tumor over background of mice in (d) and (e). Using one-tailed paired Student's t-test (n = 3 mice injected with RET₁IR@cRGD), p < 0.05 at 24 h. (h, i) NIR fluorescence imaging of urine samples (h) collected 48 h after injection from mouse in (d), and (i) from a mouse without any nanoparticle injection.

Figure 5

In vivo imaging of U87MG tumors in mice with RET₂IR@cRGD. (a, b) Time-dependent (a) in vivo bioluminescence and (b) fluorescence imaging of U87MG tumor-bearing mouse (indicated by a red arrow and circle; tumor size was about 5 mm in diameter) injected with RET₂IR@cRGD NPs (~50 μg). Acquisition time for images in (a) from left to right: 15 s (5 min), 15 s (2 h), 1 min (24 h), 1 min (48 h), and 3 min (48 h). (c) ROI analysis of the bioluminescence and fluorescence intensity between tumor and background of mice in (a) and (b). Using one-tailed paired Student's t-test (n = 3), p < 0.00002 at 5 min, and 2 h, p < 0.04 at 24 h, and p > 0.05 at 48 h. (d) In vivo bioluminescence imaging of a mouse with a small tumor of 2 mm in diameter, as indicated by a red arrow, 2 h after tail vein injection of RET₂IR@cRGD. (e) NIR fluorescence imaging of urine samples collected 48 h after injection from mice in (a). Data points represent mean±s.d. (n=4) (f-i) Histological imaging of frozen U87MG tumor slices from mouse in (a): (f) bright field, (g) NIR fluorescence (excitation filter: 480/30 nm, dichroic beamsplitter: Q570LP, emission filter: D755/40M, acquisition: 1 s), (h) Alexa Fluor 488 anti-mouse CD31 (excitation: 480/30 nm, dichroic beamsplitter: 505DCLP, emission: D535/40 nm, acquisition time: 200 ms), and (i) overlay of images in (g) and (h). Scale bar: 20 μm, objective: 20x.