Linseed Oil Nanoemulsion with Pluronic® F127 Loaded with Betulinic Acid: Preparation, Rheology, MTT Assay and in vitro Release Kinetics

The main objective of this work was to develop a nanoemulsion based on linseed oil and betulinic acid, stabilized with Pluronic F127 and polyglycerol polyricinoleate, for anticancer applications. The nanoemulsions were synthesized by ultrasound and evaluated for in vitro cytotoxicity, particle size, polydispersity index, zeta potential, morphology, encapsulation efficiency, storage stability, rheology and in vitro release kinetics. In vitro cytotoxicity assays were performed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (72 h) against HCT-116 (colorectal carcinoma), SNB-19 (glioblastoma), NCI-H460 (lung carcinoma) and L-929 (normal fibroblasts) cells. The determination of 50% inhibitory concentration (IC50) showed an increased selectivity for the emulsified betulinic acid when compared to its free form for the HCT-116 cells. The IC50 values for the synthesized nanoemulsions showed a range from 3.2 to 3.7 µM (HCT-116), 5.6 and 11.5 µM (NCI-H460), 5.8 and 7.3 µM (SNB-19) and > 16.5 µM for normal fibroblasts. In the 48 h in vitro release assays, it presented controlled release explained by the Korsmeyer-Peppas model, releasing 572.25 and 619.95 µg of betulinic acid in a controlled way, generating promising perspectives for the prolonged release of betulinic acid in anticancer applications.

本研究的核心目标是开发一种以亚麻籽油和桦木酸（betulinic acid）为基质，以泊洛沙姆F127（Pluronic F127）与聚甘油聚蓖麻油酸酯（polyglycerol polyricinoleate）为稳定剂的纳米乳剂，用于抗癌应用。该纳米乳剂通过超声法合成，并对其体外细胞毒性、粒径、多分散性指数、Zeta电位、形貌、包封率、储存稳定性、流变学特性以及体外释放动力学进行了系统评价。体外细胞毒性实验采用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法，孵育72小时后，分别针对HCT-116（结直肠癌细胞）、SNB-19（胶质母细胞瘤细胞）、NCI-H460（肺癌细胞）及L-929（正常成纤维细胞）开展检测。半数抑制浓度（IC50）测定结果显示，相较于游离态桦木酸，乳化包载的桦木酸对HCT-116细胞展现出更高的选择性。合成纳米乳剂的IC50值范围为：HCT-116细胞3.2~3.7 µM，NCI-H460细胞5.6 µM与11.5 µM，SNB-19细胞5.8 µM与7.3 µM，正常成纤维细胞则大于16.5 µM。在48小时体外释放实验中，该纳米乳剂表现出控释特性，其释放行为符合Korsmeyer-Peppas模型，以可控方式累计释放了572.25 µg和619.95 µg的桦木酸，为桦木酸在抗癌应用中的长效递送提供了颇具潜力的应用前景。