Dual-Frequency HIFU-Activated Multifunctional Nanoparticles for Trimodal Synergistic Therapy and Immune Responses with Real-Time US/MRI Guidance.

High-intensity focused ultrasound (HIFU) has been employed in clinical practice for tumor treatment via thermal ablation. However, the high energy requirements of HIFU raise safety concerns, and insufficient therapeutic efficacy can lead to potential tumor recurrence. To address these issues, we developed multifunctional theranostic nanoparticles (PFP@POMP@MnO2-Ce6 NPs) and utilized dual-frequency HIFU technology to enhance therapeutic efficiency at lower energy levels. This approach achieved efficient and rapid thermal/cavitation/sonodynamic synergistic therapy, leveraging long-term therapy through immune responses and precise monitoring via US/MRI dual-modal imaging. The NPs are composed of polymer micelles that encapsulate perfluoropentane (PFP), with their surface modified by MnO2 and sonosensitizer Chlorin e6 (Ce6). The results indicated that dual-frequency HIFU irradiation triggered the vaporization of PFP within the NPs, enhancing both the thermal and cavitation effects of HIFU, which led to an increase in the area of thermal damage in the phantoms. In vitro and in vivo experiments demonstrated that the NPs, when combined with dual-frequency HIFU, could achieve synergistic antitumor effects, inducing 94.1 ± 1.5% (p < 0.01) tumor cell death in vitro and ablation of directly treated tumors without recurrence in vivo. Most interestingly, the synergistic therapy suppressed the growth of distal tumors (93.4 ± 1.1% growth inhibition), indicating enhanced immune responses, which was further supported by immunohistochemical (IHC) results of a 69.3 ± 1.5% increase in CD8+ T cell infiltration and a 61.2 ± 1.5% increase in F4/80+ macrophage infiltration. It ensured the longevity of the treatment effects and reduced the likelihood of tumor recurrence. Moreover, the NPs' dual-modal imaging (US/MRI) capabilities provided real-time guidance and improved tumor visualization, confirming their utility as potent theranostic agents. This therapeutic approach, facilitated by multifunctional nanoparticles and in synergy with dual-frequency HIFU, provided precise imaging guidance, enhanced therapeutic efficacy, and elicited an immune response, thereby effectively addressing the limitations inherent to traditional HIFU therapy.