CLDN4-directed peptide P15-loaded nanobubbles enhance ultrasound-guided photothermal therapy and multimodal imaging in ovarian cancer models

Ovarian cancer remains a highly lethal gynecologic malignancy, largely due to the challenges of detecting and treating occult peritoneal disease, which limits complete tumor removal and image-guided interventions. Claudin 4 (CLDN4) is a protein overexpressed in epithelial ovarian carcinoma, making it a promising target for directed therapies. However, its presence in non-tumor tissues necessitates highly specific delivery systems. This study aimed to develop a novel CLDN4-directed peptide-nanobubble platform that integrates molecular recognition, imaging, and targeted therapy to overcome these limitations.

Researchers employed structure-guided in silico optimization to identify P15, a CLDN4-binding peptide. This peptide was then conjugated with indocyanine green (ICG) to form an ICG-P15 photothermal conjugate (IP). The IP conjugate was subsequently incorporated into sulfur hexafluoride-filled lipid nanobubbles, creating the IP/NBs platform. The binding affinity of P15 was validated using surface plasmon resonance. The platform's efficacy was assessed via non-permeabilized CLDN4 immunofluorescence, cellular uptake imaging, and patient-derived organoid assays. In vivo imaging studies compared IP/NBs administration with D-NBs (non-targeted dye-loaded nanobubbles) and evaluated the impact of ultrasound and near-infrared irradiation.

In silico optimization successfully identified P15 as a CLDN4-binding peptide, validated by surface plasmon resonance with a nanomolar steady-state K_D of 11.28 nM. In vitro assays confirmed CLDN4-associated P15/IP interaction and intratissue penetration in ovarian cancer models, including patient-derived organoids. The IP/NBs platform maintained both ultrasound visibility and photothermal responsiveness. Critically, ultrasound application significantly enhanced IP release and transport-related readouts. In vivo imaging demonstrated clear tumor-associated IP-related fluorescence after IP/NBs administration, which was notably higher in the IP/NBs + ultrasound group compared to the no-ultrasound group. > An IP/NBs + near-infrared irradiation group without ultrasound showed intermediate tumor suppression, while the full IP/NBs + ultrasound + near-infrared treatment achieved superior tumor suppression, highlighting the synergistic effect of targeted delivery and combined modalities.