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 <i>D</i>-limonene (<i>D</i>-LMN) nanoemulsions and to elucidate their impact on oral cancer cells. Various inhibitors, including olive oil, soybean oil, and perilla oil, were incorporated into <i>D</i>-LMN nanoemulsions at different ratios (25:75-75:25, <i>D</i>-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 <i>D</i>-LMN to inhibitor ratio produced the smallest droplet size and exhibited great stability, particularly with perilla oil. Notably, <i>D</i>-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 <i>BAX</i>, <i>BCL-XL</i>, <i>Cytochrome c</i>, and <i>Caspase-9</i>. Additionally, the nanoemulsions caused cell cycle arrest via downregulation of <i>Cyclin D1</i>, <i>CDK2</i>, <i>CDK4</i>, and <i>CDK6</i>. These findings highlight the potential of <i>D</i>-LMN nanoemulsions as a promising alternative therapeutic strategy for oral cancer treatment.