NIR‐II Excitation Phototheranostic Platform for Synergistic Photothermal Therapy/Chemotherapy/Chemodynamic Therapy of Breast Cancer Bone Metastases

Abstract To improve bone metastases treatment efficacy, current strategies are focused on the integration of chemotherapy with phototheranostic. However, the success of phototheranostic approaches is hampered by the limited tissue penetration depth of near‐infrared‐I (NIR‐I) light (700–900 nm). In this study, a NIR‐II (1000–1700 nm) excitation phototheranostic (BTZ/Fe2+@BTF/ALD) is presented for NIR‐II fluorescence imaging and NIR‐II photoacoustic imaging‐guided NIR‐II photothermal therapy (PTT), chemotherapy, and chemodynamic therapy (CDT) of breast cancer bone metastases. This phototheranostic is developed by integrating a dopamine‐modified NIR‐II absorbing donor–acceptor–donor small molecule (BBT‐FT‐DA), the boronate anticancer drug bortezomib (BTZ), and Fe2+ ions, as CDT catalysts, into an amphiphilic PEGylated phospholipid modified with the bone‐targeting ligand alendronate. In acidic and hydrogen peroxide (H2O2) over expression tumor microenvironment, the boronate–catechol linkage is cleaved and BTZ and Fe2+ ions are released to initiate the Fenton reaction, that is, chemotherapy and CDT, respectively, are initialized. It is confirmed using the murine 4T1 bone metastasis model that BTZ/Fe2+@BTF/ALD significantly suppresses the progression of tumor cells in the bone tissue via a synergistic NIR‐II PTT/chemotherapy/CDT effect. Overall, this work provides fresh insights to guide the development of NIR‐II phototheranostics for breast cancer bone metastases.

(164 mg, Yield: 56.37%). 1   was dropped into pre-prepared BTF-DA (1 ml) and stirred vigorously. After continuous agitation overnight, excess ferrous ions were removed by ultra-centrifugation at 2500 rpm for 25 min and then rinsed with water three times. Then the purified Fe 2+ @BTF-DA was diluted and filtered through a 220nm PVDF syringe driven filter, and the Fe 2+ @BTF-DA stock solution was obtained by ultra-centrifugation.
The preparation method was referred to Fe 2+ @BTF-DA.  In vitro Cellular Uptake. The 4T1 cells were used for the assessment of cellular uptake. First, cells, seeded 4 × 10 4 per well, were incubated in DMEM in a 6-well plate at 37 °C and 5% CO 2 condition for 24 h. Subsequently, the medium was replaced by one of the two groups, including 1 mL fresh DMEM medium alone and the 1mL mixture of fresh DMEM medium and BTZ/Fe 2+ @BTF-DA (0.1 mg mL -1 ), respectively. The treated cells were further incubated at 37 °C and 5% CO 2 condition for 4 h. Afterward, the upper supernatant was sucked out and 1 mL of PBS was added to clean twice and remove dead cells. Subsequently, PBS was removed and 500 μL trypsin digestion solution with EDTA was added, and the cells were dissolved in an incubator at 37 °C and 5% CO 2 for 90 s. Later 1 mL of DMEM was added and the cells were transferred into a 15 mL centrifuge tube and centrifuged for 3 min. The supernatant was then removed, and 100 μL of PBS was added to the centrifuge tube. Finally, the cells were transferred to a 96-well plate, where NIR-II fluorescence images of the two groups were captured under the 1064 nm laser excitation.

Cellular internalization. To track internalization of BTZ/Fe 2+ @BTF-DA in tumor cells, 4T1
cells (1 × 10 4 ) were grown on slices in a 6-well culture plate at 37 °C and 5% CO 2 condition for 24 h, and then incubated with FITC/BTZ/Fe 2+ @BTF-DA (0.1 mg mL -1 ) . FITC was used as a fluorescent marker to determine whether the material was uptaken by cells. After 4 h, the cells were washed with PBS for three times, followed by costaining with DAPI (nucleus indicator, 10 min). Then, the cells were imaged by . image was performed using the NIR-II in vivo imaging system software.
Mice are mounted on specially designed holders, which rotate 360 degrees with sophisticated rotating motors. Four short pulsed laser beams (biorthogonal, bioblique) are emitted by a laser the tissue absorbs light energy to generate ultrasonic signal, which is received by a professionally designed 125° are transducer. The signal was collected at 360°, and the photoacoustic image was reconstructed by special 3D reconstruction software through unit transformation of DAQ data.
Bone targeting assay. Two mice were injected into the tail vein with BTZ/Fe 2+ @BTF-DA (as a control group) and BTZ/Fe 2+ @BTF/ALD, the real-time in vivo NIR-II fluorescence imaging was performed at different post-injection times by using an in vivo NIR-II fluorescence imaging system. The analysis of the signal intensity of NIR-II image was performed using the NIR-II in vivo imaging system software.

4T1
bone metastasis models mice