Bioorthogonally activatable cyanine dye with torsion-induced disaggregation for in vivo tumor imaging

Abstract

Advancement of bioorthogonal chemistry in molecular optical imaging lies in expanding the repertoire of fluorophores that can undergo fluorescence signal changes upon bioorthogonal ligation. However, most available bioorthogonally activatable fluorophores only emit shallow tissue-penetrating visible light via an intramolecular charge transfer mechanism. Herein, we report a serendipitous "torsion-induced disaggregation (TIDA)" phenomenon in the design of near-infrared (NIR) tetrazine (Tz)-based cyanine probe. The TIDA of the cyanine is triggered upon Tz-transcyclooctene ligation, converting its heptamethine chain from S-trans to S-cis conformation. Thus, after bioorthogonal reaction, the tendency of the resulting cyanine towards aggregation is reduced, leading to TIDA-induced fluorescence enhancement response. This Tz-cyanine probe sensitively delineates the tumor in living mice as early as 5 min post intravenous injection. As such, this work discovers a design mechanism for the construction of bioorthogonally activatable NIR fluorophores and opens up opportunities to further exploit bioorthogonal chemistry in in vivo imaging.

Fig. 1. Strategies for Tz-based bioorthogonally activatable probes.

Tz-quenching mechanism (a) and the torsion-induced disaggregation (TIDA) for activatable NIR Tz-cyanine in our work (b).

Fig. 2. Molecular design and spectra properties of cyanines.

Chemical structures of a CyP7, b bioorthogonal-activated cyanines, and c control cyanines. Bioorthogonal-moiety Tz (red color group) with methyl (shorter) (CyP7T) or PEG3 (longer) (CyP7N) distances from substrate structures. CyP7TT/CyP7NT activated by CyP7T/CyP7N and TCO via IEDDA reaction. d Normalized absorption (black)/emission (red) spectra of dyes. e Fluorescence intensity at the maximum emission spectra with different concentrations of cyanines. f–h Fluorescence loss with concentrations. Dotted lines represent ideal linear relationship between fluorescence intensity and concentration, indicating non-quenching in blue range. i ACQ effect for control group (CyP7NT formed by CyP7N and TCO).

Fig. 3. Mechanism for TIDA effect under theoretical calculations.

a Representative molecular structures and conformations of CyP7, CyP7-CH, CyP7T, and CyP7TT cyanines optimized at B3LYP/6-31G* level (hydrogen atoms, N-substituents on benzoindole rings and meso-amino-substituents are neglected for clear vision). b HOMO/HOMO-1/LUMO orbitals and energies with the largest oscillator strengths for CyP7, CyP7-CH, CyP7T, and CyP7TT (hydrogen atoms are neglected).

Fig. 4. ¹H and 2 D ROESY NMR analysis.

¹H NMR shift changes of vinyl-hydrogens (H₁, H₂, H₃, H₄) for a CyP7, b CyP7T, c CyP7TT, d CyP7N, and e CyP7NT. Orange regions refer to H₁/H₄ and blue regions refer to H₂/H₃. 2D ROESY NMR of f CyP7T and g CyP7TT in DMSO-d₆.

Fig. 5. Bioorthogonal imaging of CyP7T in response to TCO in 4T1 cells.

a CLSM imaging of 4T1 cells pre-incubated with different concentrations of TCO (0, 5, 10, 20 μM) for 3 h, washed with PBS, followed by incubation with CyP7T (5 μM) for 0.5 h. Cell nuclei were stained with DAPI (blue). b Qualitative analysis of fluorescence in Fig. 5a. Data are presented as mean values ± SD. A two-sided student’s t-test was performed (1:1 vs 0:1, p = 0.0244; 2:1 vs 1:1, p = 0.0102; 2:1 vs 0:1, p = 0.0015; 4:1 vs 2:1, p = 0.2988). c Confocal fluorescence images of 4T1 cells pre-incubated with 10 μM TCO for 3 h, washed with PBS, followed by incubation with 5 μM CyP7T or CyP7N for 5, 10, 15, 30 min. d Qualitative analysis of fluorescence in Fig. 5c. Data are presented as mean values ± SD. A two-sided student’s t test was performed (p = 0.2205, 0.1959, 0.0139, 0.5054, 0.0025, 0.2600, 0.0033, 0.9434, 0.0188, and 0.8194 from left to right). Fluorescence images of cells were recorded at λ_ex = 405 nm for DAPI, λ_ex = 640 nm for NIR dyes. Scale bar: 30 μm. Error bars: standard deviation from three separate measurements. *p < 0.05, **p < 0.01, ns not significant.

Fig. 6. Bioorthogonal-activated imaging in 4T1 cells and MCF-7 cells.

a CLSM imaging of 4T1 cells (integrin α_vβ₃ high-expressing) incubated with CyP7T, CyP7T/TCO, and CyP7T/TCO-RGD (5 μM) for 0.5 h. b Timeline for the addition of CyP7T and TCO/TCO-RGD. Cells were pre-incubated with 10 μM TCO or TCO-RGD for 3 h, washed with PBS, followed by incubation with CyP7T (5 μM) for 10 min. c Quantification of NIRF signal in a. Data are presented as mean values ± SD. A two-sided student’s t test was performed (CyP7T/TCO-RGD vs CyP7-CH, p = 0.0003; CyP7T/TCO-RGD vs CyP7T, p = 0.0006; CyP7T/TCO-RGD vs CyP7T/RGD, p = 0.0017). d CLSM imaging of MCF-7 cells (integrin α_vβ₃ low-expressing) incubated with CyP7T, CyP7T/TCO, and CyP7T/TCO-RGD (5 μM) for 0.5 h. e Quantification of NIRF signal in d. Data are presented as mean values ± SD. A two-sided student’s t test was performed (CyP7T/TCO vs CyP7T, p = 0.0187; CyP7T/TCO vs CyP7T/TCO-RGD, p = 0.8359). Cells were stained with nuclear dye, DAPI (blue). All the images were acquired at 40× magnification. Scale bar: 30 μm. Error bars: standard deviation from three separate measurements. **p < 0.01, ns not significant.

Fig. 7. In vivo bioorthogonal NIR imaging for 4T1 tumor-bearing mice.

a Timeline of RGD-TCO, CyP7T injection, and NIR imaging. Mice were pre-injected with TCO-RGD (10 nmol) or saline, followed by injection of CyP7T (5 nmol) 12 h later. b NIR imaging was performed at various timepoints (0, 0.5, 1, 2, 4, 8, 12, 24, 48 h) post-injection of CyP7T. c Fluorescence signal intensity in tumor tissue and d Tumor-to-background ratio (TBR) over time, n = 3. TBR was determined by (tumor ROI—background ROI)/background ROI. e Fluorescence intensity analysis of excised organs harvested from mice 24 h post-injection of CyP7T. Data are presented as mean ± SD. (n = 3 biologically independent mice per group).