Development of D-Limonene Nanoemulsions for Oral Cancer Inhibition: Investigating the Role of Ostwald Ripening Inhibitors and Cell Death Mechanisms.

The aim of this study was to investigate the effect of Ostwald ripening inhibitors on D-limonene (D-LMN) nanoemulsions and to elucidate their impact on oral cancer cells. Various inhibitors, including olive oil, soybean oil, and perilla oil, were incorporated into D-LMN nanoemulsions at different ratios (25:75-75:25, D-LMN to inhibitor). The resulting nanoemulsions were evaluated for droplet size, size distribution, zeta potential, stability, droplet morphology, cytotoxicity, antimetastatic and anti-invasive activities, apoptosis induction, and cell cycle arrest. Results showed that the 75:25 D-LMN to inhibitor ratio produced the smallest droplet size and exhibited great stability, particularly with perilla oil. Notably, D-LMN nanoemulsions displayed strong anti-oral cancer effects by reducing cell viability, metastasis, and invasion. Apoptosis was induced, as evidenced by nuclear fragmentation, Annexin V binding, and altered expression of BAX, BCL-XL, Cytochrome c, and Caspase-9. Additionally, the nanoemulsions caused cell cycle arrest via downregulation of Cyclin D1, CDK2, CDK4, and CDK6. These findings highlight the potential of D-LMN nanoemulsions as a promising alternative therapeutic strategy for oral cancer treatment.