Abstract: Based on previous research suggesting that milk exosomes specifically express miR-26a, this study optimized the miR-26a encapsulation efficiency of liposomes through ultrasonic treatment, and it also evaluated the in vitro digestion performance of the ultrasonic-treated liposomes and compared its anti-inflammatory activity with that of natural milk exosomes. Single-factor experiments were conducted to optimize the molar charge ratio of cationic liposome vector to nucleic acid (N/P), ultrasonication time, and ultrasonic power for the preparation of miR-26a-encapsulated liposomes. The results demonstrated that when the N/P ratio was 4, ultrasonication time was 15 min, and ultrasonic power was 300 W, the encapsulation efficiency reached its peak of (89.51 ± 1.37)%, with uniform particle size distribution and homogeneous morphology. In vitro simulated digestion experiments showed that the retention rate of miR-26a in the ultrasonic-treated liposomes (58%) was higher than that in the conventional liposomes (24%). Cell function experiments confirmed that the ultrasonic-treated liposomes not only enhanced the viability of normal Caco-2 cells but also effectively inhibited lipopolysaccharide (LPS)-induced inflammatory responses, significantly restoring cell viability under inflammatory conditions. Additionally, it reduced NO release and down-regulated the secretion of the inflammatory cytokines interleukin 6 (IL-6), tumor necrosis factor α, and IL-1β, while effectively controlling the generation of reactive oxygen species. Notably, the ultrasonic-treated liposomes showed no significant differences from milk exosomes in terms of cell viability regulatory and inflammation inhibitory effects. In summary, this study demonstrates that ultrasonic can significantly improve the miR-26a delivery efficiency of liposomes.

Key words: miR-26a; ultrasound; liposomes; bovine milk exosomes; anti-inflammatory activity

摘要： 基于牛乳外泌体特异性表达miR-26a的研究基础，通过超声工艺优化脂质体包封率，同时考察其体外消化性能，以及与天然牛乳外泌体抗炎活性差异。采用单因素试验优化阳离子脂质载体与核酸电荷物质的量比（N/P值）、超声时间及超声功率，制备miR-26a脂质体。结果表明：当N/P值为4、超声时间15 min、超声功率300 W时，脂质体包封率达峰值（（89.51±1.37）%），粒径分布均匀且形态均一。体外模拟消化实验结果显示，超声脂质体组的miR-26a保留率（58%）高于常规脂质体组（24%）。细胞功能实验证实，超声优化处理在提升正常Caco-2细胞活力的同时，还可有效抑制脂多糖诱导的炎症反应，使炎症状态下的细胞活力得到明显恢复，同时降低NO释放量，并下调炎症因子白细胞介素6（interleukin 6，IL-6）、肿瘤坏死因子α及IL-1β分泌，活性氧的生成亦得到有效控制。超声脂质体在调节细胞活力及炎症抑制效应方面与牛乳外泌体无显著差异。综上，本研究证实超声技术可显著提高miR-26a脂质体的递送效能。

关键词: miR-26a；超声；脂质体；牛乳外泌体；抗炎活性