Revolutionizing drug delivery: low-intensity pulsed ultrasound (LIPUS)-driven deep penetration into hypoxic tumor microenvironments of cholangiocarcinoma.

Background: Hypoxia is a major obstacle in the treatment of solid tumors because it causes immune escape and therapeutic resistance. Drug penetration into the hypoxic regions of tumor microenvironment (TME) is extremely limited. This study proposes using the unidirectional fluid flow property of low-intensity pulsed ultrasound (LIPUS) to overcome drug penetration limitations in the TME. LIPUS is gaining attention as a therapeutic modality for cancer owing to its safety and efficacy. Methods: LIPUS parameters, such as the intensity, duty cycle (DC), and duration, were optimized to enhance drug delivery into the hypoxic regions of the TME in cholangiocarcinoma (CCA). Transparent tumor imaging using the tissue optical clearing method (CLARITY) enabled 3D visualization and quantitative assessment of drug delivery and therapeutic efficacy in relation to blood vessels in an intact tumor at the micrometer level. The antitumor efficacy of LIPUS-assisted chemotherapy was evaluated in a CCA xenograft mouse model. Results: LIPUS significantly enhanced drug delivery efficacy into the hypoxic region of the TME in CCA. Under optimal conditions, i.e., a DC of 45% and a spatial-peak temporal-average intensity (Ispta) of 0.5 W/cm², drug penetration, including liposomal nanoparticles and chemotherapeutic agents gemcitabine and cisplatin, was improved by approximately 1.8-fold, resulting in a fivefold increase in apoptotic cancer cell death and a significant reduction in CCA growth. Notably, drug penetration and efficacy were more significantly affected by DC compared to the spatial-peak pulse-average intensity (Isppa). The efficacy saturated at Ispta values above 0.5 W/cm² under a 45% DC. Furthermore, we confirm that LIPUS induces non-thermal effects without causing cell damage, ensuring biosafety. These findings highlight the potential of LIPUS as a non-invasive strategy for treating hypoxic tumors. Conclusion: LIPUS adjuvant therapy promises improved cancer treatment outcomes and offers a safe and innovative therapeutic strategy for CCA and other hypoxic tumors.