To prepare CaCO₃ nanoprobes loaded with polydopamine (PDA) of pH-responsive diagnosis and treatment, and evaluate their effects in ultrasonic/photoacoustic dual-mode imaging and photothermal therapy of breast cancer in vitro.

PEG-modified CaCO₃ nanoprobes (CaCO₃-PDA-PEG) were prepared by one-pot gas phase diffusion method and two-step method. The macroscopic and microscopic morphology, particle size, infrared spectrum, stability, and other characteristics of CaCO₃-PDA-PEG nanoprobes were evaluated. The ultrasonic/photoacoustic dual-mode imaging effect and pH-responsive CEUS imaging ability of the nanoprobes were detected. Then, we evaluated the photothermal conversion efficiency and cytotoxicity of the nanoprobes. Also, the tumor cell targeting ability was assessed by confocal microscopy. Finally, cell proliferation assay and live and dead cell staining were used to evaluate the tumor killing efficiency of the nanoprobes in vitro.

We prepared the CaCO₃-PDA-PEG nanoprobes successfully. These spherical nanoprobes were uniform in size, with an average particle size of ~258 nm and a surface potential of ~-21 mV. Besides, the nanoprobes could obviously enhance the photoacoustic imaging signal with the increase of the concentration. Furthermore, CaCO₃-PDA-PEG nanoprobes could produce CO₂ bubbles when exposed to H⁺ in the acidic tumor microenvironment (pH 6.5 and pH 5.5), which enhanced CEUS imaging signal. Confocal laser scanning microscopy showed that the nanoprobes could target tumor cells efficiently in a passive way. When incubated with 4T1 cells, these nanoprobes (4 mg/ml) showed a negligible cytotoxicity as the cell survival rate was as high as 92.31%. Yet, once irradiated by an 808 nm laser, CaCO3-PDA-PEG nanoprobes had an obvious photothermal killing effect. The tumor cell survival rate decreased to 26.61% and there was a large amount of red fluorescence (dead cells) under confocal laser scanning microscope.

The prepared pH-responsive CaCO₃-PDA-PEG nanoprobes could significantly enhance the ultrasonic/photoacoustic dual-mode imaging signal and photothermal therapy efficacy for tumors in vitro, laying a foundation for the further development of accurate visualized photothermal therapy for tumors.