Background: The integration of NIR photothermal therapy and chemotherapy is considered as a promising technique for future cancer therapy. Hollow Prussian nanospheres have attracted much attention due to excellent near-infrared photothermal conversion effect and drug-loading capability within an empty cavity. However, to date, the hollow Prussian nanospheres have been prepared by a complex procedure or in organic media, and their shell thickness and size cannot be controlled. Thus, a simple and controllable route is highly desirable to synthesize hollow Prussian nanospheres with controllable parameters. Materials and Methods: Here, in our designed synthesis route, the traditional FeCl 3 precursor was replaced with Fe 2 O 3 nanospheres, and then the Prussian blue (PB) nanoparticles were engineered into hollow-structured PB (HPB) nanospheres through an interface reaction, where the Fe 2 O 3 colloidal template provides Fe 3+ ions. The reaction mechanism and control factors of HPB nanospheres were systematically investigated. Both in vitro and in vivo biological effects of the as-synthesized HPB nanospheres were evaluated in detail. Results: Through systematical experiments, a solvent-mediated interface reaction mechanism was put forward, and the parameters of HPB nanospheres could be easily adjusted by growth time and template size under optimal water and ethanol ratio. The in vitro tests show the rapid and remarkable photothermal effects of the as-prepared HPB nanospheres under NIR laser irradiation (808 nm). Meanwhile, HPB nanospheres also demonstrated a high DOX loading capacity of 440 mg g −1 as a drug carrier, and the release of the drug can be regulated by the heat from PB shell under the exposure of an NIR laser. The in vivo experiments confirmed the outstanding performance of HPB nanospheres in photothermal/ chemo-synergistic therapy of cancer. Conclusion: A solvent-mediated template route was developed to synthesize hollow Prussian blue (HPB) nanospheres in a simple and controllable way. The in vitro and in vivo results demonstrate the as-synthesized HPB nanospheres as a promising candidate due to their low toxicity and high efficiency for cancer therapy.

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