Ultrasound-Targeted Nanobubbles Codelivering NKP-1339 and miR-142-5p for Synergistic Mitochondrial Immunogenic Cell Death and PD-L1 Inhibition in Cancer Therapy

The combination of chemical immunotherapy and gene therapy holds great promise for malignant tumor treatment. Here, we developed an ultrasound-targeted Liposome nanobubbles system (NKP-1339/miR-142-NBs) for precise codelivery of drugs and genes to treat esophageal squamous cell carcinoma (ESCC) with ultrasound-targeted microbubble destruction (UTMD). This study systematically investigated the system's therapeutic mechanisms-including mitochondrial dysfunction induction, immunogenic cell death (ICD), and antitumor immune activation-alongside its pharmacokinetics and targeting efficiency. In an ESCC mouse model, NKP-1339/miR-142-NBs combined with ultrasound markedly suppressed tumor growth (79.72% ± 0.1% vs. NB control 18.79% ± 1.29%) through NKP-1339 triggering ICD and miR-142-5p down-regulating programmed death-ligand 1 (PD-L1) expression, synergistically potentiating immune responses. Furthermore, we found that triggering ICD, including the exposure of calreticulin on the cell membrane, was related to altering mitochondrial fission dynamics in the ESCC cells. The down-regulation of PD-L1 expression by miR-142-5p reactivated CD8⁺ T cells by relieving programmed death-1 (PD-1)/PD-L1-mediated immunosuppression, enhancing immune memory and antitumor efficacy. Moreover, the UTMD technique enhanced the tumoral accumulation and penetration of nanobubbles, improving delivery specificity and minimizing off-target effects. This combined treatment strategy, including UTMD, provides a promising translational potential for ESCC therapy.