Cavitation-Free Acoustic Sensitization Enhances PLK4-Targeted Therapy Using the Phillyrin Derivative DE02 in Osteosarcoma

Abstract

Objective:

To ascertain whether cavitation-free acoustic sensitization enhances intracellular delivery and amplifies polo-like kinase 4 (PLK4)-targeted signaling to boost the phillyrin derivative DE02’s antitumor efficacy against osteosarcoma, providing a secure and effective ultrasound-enabled method for targeted cancer biotherapy.

Methods:

Using ultracentrifugation, exosomes generated from human umbilical vein endothelial cells were separated, purified, and identified using common morphological and molecular markers. Low-energy sonication under cavitation-free acoustic circumstances was used to insert DE02 into exosomes, creating an exosomal delivery (ExoDE02) system intended to improve cellular absorption without causing membrane damage. MG-63 and Saos-2 human osteosarcoma cell lines were used as in vitro models. The cell counting kit-8 test was used to measure cell proliferation, proliferating cell nuclear antigen (PCNA) immunofluorescence was used to measure proliferative activity, and Transwell assays were used to measure migration and invasion. Enzyme-linked immunosorbent assay (ELISA), real-time quantitative PCR, and Western blotting were used to assess the expression of PLK4 and downstream tumor protein 53 (p53)–cyclin-dependent kinase inhibitor 1A (p21) signaling components. To verify pathway specificity, PLK4 overexpression studies were carried out. A nude mouse xenograft model was used to evaluate in vivo antitumor effectiveness and biosafety.

Results:

In a concentration-dependent manner, DE02 showed almost 10 times more antiproliferative action against osteosarcoma cells than the parent chemical phillyrin. The inhibitory effects of DE02 on osteosarcoma cell proliferation, migration, and invasion were greatly enhanced by cavitation-free acoustic sensitization-mediated ExoDE02. This was accompanied by a significant downregulation of PCNA and PLK4 expression and activation of the p53–p21 tumor suppressor pathway. The anticancer effects of DE02 and ExoDE02 were successfully inhibited by overexpression of PLK4, indicating PLK4-dependent therapeutic efficacy. ExoDE02 significantly inhibited the growth of xenograft tumors in vivo, decreased tumor weight and volume, and showed no discernible systemic toxicity.

Conclusions:

By increasing the therapeutic efficacy of the phillyrin derivative DE02 via a safe, nondestructive exosome-based delivery method, cavitation-free acoustic sensitization improves PLK4-targeted osteosarcoma therapy. For targeted osteosarcoma biotherapy, this ultrasound-enabled method provides a mechanistically defined and physiologically applicable platform.