Modular Design of High-Brightness pH-Activatable Near-Infrared BODIPY Probes for Noninvasive Fluorescence Detection of Deep-Seated Early Breast Cancer Bone Metastasis: Remarkable Axial Substituent Effect on Performance

Abstract

We herein report a series of high-brightness pH-activatable near-infrared (NIR) BODIPY probes for high-contrast intravital imaging of deep-seated early breast cancer bone metastasis by harnessing the axial substituent effect. These probes exhibit tunable pK _a, higher brightness, and antiquenching capabilities in aqueous solution, which can be simultaneously adjusted by axial steric substituents. The optimized probe BODO-3 bearing axial dimethyl substituents exhibited a higher pK _a value of 5.6 and a brighter NIR fluorescence under tumor acidic pH, showing 10.3-fold and 6.5-fold enhanced brightness (εΦ) at pH 5.5 and 6.5, respectively. Due to the higher brightness, BODO-3 with a brilliant NIR emission at 700 nm allows for deep optical penetrations of 5 and 8 mm at pH 6.5 and 4.5, respectively. Meanwhile, covalent functionalization with glucose (BODO-3-Glu) could further enhance breast cancer and its soft tissue metastasis imaging in vivo. Notably, covalent functionalization with bisphosphonate (BODO-3-PO ₃ H ₂ ) allowed the successful targeting and visualization of deep-seated bone metastases of breast cancer with a high tumor to normal contrast of 8/1, outperforming X-rays in early detection. This strategy may provide insights for designing high-brightness activatable NIR probes for detecting deep-seated tumors and metastases.

Figure 1

Schematic illustration of the modular design of high-brightness pH-activatable NIR BODIPYs by harnessing an axial substituent effect. Deep-tissue bioimaging was achieved using these probes, and covalent functionalization with glucose and bisphosphonate further improved intravital imaging of subcutaneous breast cancer and deep-seated bone metastases, respectively.

Figure 2

(a) Synthesis of a series of pH-activatable NIR BODIPY probes with different axial substituents. (b) Photophysical data of key probes at pH 5.5.

Figure 3

Systematic evaluation of axial substituent effects on NIR BODIPY probes. (a) Normalized absorption and emission spectra of BODO-3 (5 μM) in citrate buffer (pH 4.0). (b) NIR fluorescence spectra of BODO-3 (5 μM) in different pH buffers. (c) Normalized pH titration profiles of the NIR BODIPY probes. pK_a: BOD-1, 4.6; BOD-2, 5.6; BOD-3, 5.0; BODO-1, 4.6; BODO-2, 5.1; BODO-3, 5.6; BODO-4, 5.3; BODO-5, 4.9; BODO-6, 4.0. (d) Fluorescence images of probes (5 μM) in different pH buffers. (e, f) Normalized pH-dependent fluorescence intensity of probes BOD to BOD-1 (e) and BODO to BODO-1 (f) (5 μM). (g, h) Relative fluorescence intensity of the BOD (g) and BODO (h) series probes (5 μM) in the absence or presence of 10% FBS. (i) Fluorescence emission spectra of probes (5 μM) in different solvents (red, THF; blue, EtOH; purple, H₂O) with 0.5% (v/v) trifluoroacetic acid (TFA). BOD and BODO series probes were excited at 640 and 660 nm, respectively.

Figure 4

High-brightness pH-activatable NIR BODO-3 enables deep-tissue imaging ex vivo and in vivo. (a) NIR fluorescence images of probes (10 μM) covered with different thicknesses of chicken breast slices in acidic buffers. (b) Relative ratios of fluorescence intensities in (a). (c) In vivo fluorescence images of mice after intramuscular injection of 60 μL (1 μM, pH 5.6 citrate buffer) of BODO-1 and BODO-3 in the left and right thighs, respectively, covered by chicken breast tissue with varying thicknesses. (d) In vivo fluorescence images of mice bearing subcutaneous 4T1 breast cancer xenograft tumors after an intratumor injection of 60 μL (1 μM, pH 7.4 PBS) of BODO-1 and BODO-3 in the left and right tumors, respectively, covered by chicken breast tissue with varying thicknesses. (e) Relative (BODO-3/BODO-1) ratios of fluorescence intensities in the muscles and tumors using ROI analysis (n = 3). (f) In vivo bioluminescence and fluorescence images of mice bearing metastatic breast cancer tumors after i.v. injection of 2.0 nmol (10 μM, 200 μL PBS) of BODO-3. (g) Representative ex vivo bioluminescence and fluorescence images of harvested organs at 24 h postinjection.

Figure 5

Covalent attachment of glucose and bisphosphonate groups onto BODO-3 can further enhance tumor imaging. (a) Synthesis of functionalized BODO-3-Glu, BODO-3-Gal, and BODO-3-PO₃H₂. (b) BODO-3-PO₃H₂ specifically bound to HA and remained pH-responsive: (i) suspensions of HA with 10 μM BODO-3 (α), BODO-3-CO₂H (β), BODO-3-PO₃H₂ (γ), BODO-3-Gal (δ), and BODO-3-Glu (ε) in water; (ii, iii) NIR fluorescence images of the corresponding diluted suspensions in pH 5.0 buffer (ii) and pH 7.4 buffer (iii). (c) Time-dependent fluorescence images of mice bearing subcutaneous 4T1 breast cancer tumors after i.v. injection of 2.0 nmol (10 μM, 200 μL) probes. Representative fluorescence images of harvested tumors and organs at 24 h postinjection. (d) Normalized time-dependent fluorescence intensities on the subcutaneous 4T1 tumors by ROI analysis (n = 2). (e) Tumor to organ (liver, kidneys, and muscle) ratios of relative probes.

Figure 6

BODO-3-PO₃H₂ can sensitively detect deep-seated early breast cancer bone metastasis. (a) Time-dependent in vivo X-ray, bioluminescence, and fluorescence images of mice bearing bone metastases of 4T1 breast cancer after i.v. injection of 2.0 nmol (10 μM, 200 μL) of BODO-3-PO₃H₂ and BODO-3-Glu, respectively. Arrowheads: bone metastasis (yellow); spread of tumor (purple). (b) Bioluminescence and fluorescence images of harvested femurs and organs from 4T1 bone metastasis bearing mice sacrificed at 24 h postinjection. Abbreviations: He, heart; Li, liver; Lu, lung; Ki, kidneys; Sp, spleen; Pa, pancreas; Mu, muscle. (c) Bioluminescence and fluorescence images of harvested femurs and organs from 4T1 bone metastasis bearing mice at the late stage sacrificed at 24 h postinjection. The left hindlimb (i) was separated into two parts of left femur and resected metastasis in soft tissues (ii). Abbreviations: RF, right femur; LF, left femur. (d) Femur to organ ratios of BODO-3-PO₃H₂ and BODO-3-Glu fluorescence intensity by ROI analysis at 24 h postinjection. (e) Tumor to organ ratios of BODO-3-Glu fluorescence intensity by ROI analysis at 24 h postinjection. (f) White light images of a normal femur and a metastatic femur: histology and fluorescence microscopy analysis of BODO-3-PO₃H₂ accumulation and activation in normal femur and femur metastases. BODO-3-PO₃H₂ is shown in red. Scale bar: 100 μm.