In silico design of CLDN4-directed peptide-loaded nanobubbles for ultrasound-assisted delivery, photothermal therapy, immune activation, and multimodal imaging in ovarian cancer.

BACKGROUND: Ovarian cancer remains among the most lethal gynecologic malignancies, partly because occult peritoneal disease limits complete macroscopic cytoreduction and image-guided intervention. Claudin 4 (CLDN4) is enriched in epithelial ovarian carcinoma but is not tumor-exclusive; therefore, rational CLDN4-directed delivery requires consideration of tumor enrichment, surface accessibility, and ligand engagement. This study aimed to develop a CLDN4-directed peptide-nanobubble phototheranostic platform integrating molecular recognition, ultrasound/fluorescence imaging, ultrasound-associated IP-related delivery/retention, and photothermal treatment.

RESULTS: Structure-guided in silico optimization and surface plasmon resonance validation identified P15 as a CLDN4-binding peptide with nanomolar affinity (steady-state K_D = 11.28 nM). P15 was conjugated with indocyanine green (ICG) to generate an ICG-P15 photothermal conjugate (IP), which was incorporated into sulfur hexafluoride-filled lipid nanobubbles to form IP/NBs. Surface-preserving binding and competition assays, non-permeabilized CLDN4 immunofluorescence, cellular uptake imaging, and patient-derived organoid assays supported CLDN4-associated P15/IP interaction and intratissue penetration in the tested ovarian cancer models. IP/NBs preserved ultrasound visibility and photothermal responsiveness and showed ultrasound-associated changes in IP release and transport-related readouts under the applied acoustic conditions. In vivo imaging demonstrated tumor-associated IP-related fluorescence after IP/NBs administration, with D-NBs serving as a non-targeted dye-loaded nanobubble comparator. Matched post-ultrasound imaging further showed higher tumor-associated fluorescence in the IP/NBs + ultrasound group than in the no-ultrasound group, and an IP/NBs + near-infrared irradiation group without ultrasound showed intermediate tumor suppression compared with the full IP/NBs + ultrasound + near-infrared treatment. The full treatment regimen produced marked tumor suppression and was associated with increased oxidative-stress readouts, calreticulin exposure, ATP/HMGB1 release, cytokine secretion, immune-cell activation, and tumor-associated T-cell infiltration.

CONCLUSIONS: This study develops and evaluates a CLDN4-directed, IP-containing nanobubble framework for ovarian cancer phototheranostics. The data support ultrasound-associated enhancement of tumor IP-related delivery/retention, photothermal tumor suppression, and treatment-associated immune activation under the tested workflow, while defining areas for further optimization of peptide stability, nanobubble pharmacology, acoustic dosing, and translational safety.