Tocotrienols (T3) have been known to have potential cytotoxic activity against cancer cells. However, their ability to effectively target cancerous tissue is hampered by their lipophilic characteristics and poor bioavailability. In this study, encapsulating T3 into nanoemulsions (NE) prepared from palm oil (5-20%) and Tween-80 (3-7%) assisted with ultrasonication (5-20 min) resulted in improved stability of NE-T3 (kinetic, thermodynamic, long-term storage) and biological activity. Response Surface Methodology with Box-Behnken Design predicted the significant effect of palm oil concentrations on nanosized particle formation, while Tween-80 concentration and ultrasonication time had minimal effect on particle size. The optimum formulation achieved nanoscale characteristics of NE-T3, including a mean particle size of 64.8 ± 8 nm, polydispersity index of 0.288, and ζ-potential of - 19.16 mV, with only 5.2 ± 0.8% size variation after 60-day storage at 40 °C. It also demonstrated condition-specific thermodynamic stability, long-term storage capability, controlled drug release properties (77.97 ± 4.41% cumulative release over 54 h), and significantly enhanced antioxidant activity. The antioxidant activity (ABTS and DPPH) of NE-T3 showed IC₅₀ values of 26.63 ± 1.8 µg/mL (ABTS) and 9.38 ± 1.24 µg/mL (DPPH), representing 1.5-2.0-fold improvement over free form of T3. The NE-T3 significantly enhanced cytotoxicity (IC₅₀ 45.07 ± 4.84 µg/mL) compared to free-T3 (IC₅₀ 78.75 ± 4.68 µg/mL) in B16F0 melanoma cells, while both showed minimal toxicity on normal NIH-3T3 cells (IC₅₀ > 230 µg/mL). Additional anticancer assays revealed that NE-T3 induced 12.83 ± 2.1% late apoptosis and caused G1 phase cell cycle arrest. This study highlights the potential of NE-T3 as a promising platform for r combinatory cancer therapies.

Keywords: Antioxidant; Antiproliferative; Apoptosis; Nanoemulsion; Stability; Tocotrienol.

© 2025. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.

生育三烯酚（T3）已被证实具有对抗癌细胞的潜在细胞毒性作用。然而，由于其亲脂性特性和较差的生物利用度，它们有效靶向癌组织的能力受到了限制。在这项研究中，将T3封装到用棕榈油（5-20%）和Tween-80（3-7%）制备的纳米乳液（NE）中，并通过超声波处理（5-20分钟），改善了NE-T3（动力学、热力学、长期储存）的稳定性和生物活性。响应面法结合Box-Behnken设计预测了棕榈油浓度对纳米级颗粒形成的重要影响，而Tween-80浓度和超声时间对粒径的影响较小。最佳配方使NE-T3达到了纳米特征特性，包括平均粒径64.8±8nm、多分散指数0.288以及ζ电位-19.16mV，在40℃下储存60天后粒径变化仅为5.2±0.8%。它还显示了条件特定的热力学稳定性、长期存储能力、控释性质（54小时内累计释放77.97±4.41%）以及显著增强的抗氧化活性。NE-T3的抗氧化活性（ABTS和DPPH）IC₅₀值分别为26.63±1.8 µg/mL（ABTS）和9.38±1.24 µg/mL（DPPH），这比游离形式的T3提高了1.5-2.0倍。NE-T3在B16F0黑色素瘤细胞中显著增强了细胞毒性（IC₅₀ 45.07±4.84 µg/mL），而游离T3为（IC₅₀ 78.75±4.68 µg/mL），两者对正常NIH-3T3细胞的毒性均较低（IC₅₀>230 µg/mL）。额外的抗癌试验表明，NE-T3诱导了12.83±2.1%晚期凋亡并导致G1期细胞周期停滞。本研究突出了NE-T3作为组合癌症疗法平台的潜力。

关键词：抗氧化；抗增殖；凋亡；纳米乳液；稳定性；生育三烯酚。

© 2025. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.