番茄外泌体的分离提取工艺优化及其作为药物载体的可行性分析

目的 优化番茄外泌体提取工艺,初步探索番茄外泌体作为纳米载体的可行性。方法 利用蔗糖密度梯度法提取番茄外泌体;动态光散射仪和透射电镜鉴定外泌体纳米粒子特征;用MTT 法检测外泌体对A549、A2780 及HONE1（EBV+）3 种肿瘤细胞的毒性。构建外泌体携带模型药物DiI 和目的基因反义EBV-miR-BART7-3p 的纳米药物,考察其细胞摄取能力,实时荧光定量聚合酶链反应检测基因药物的沉默效果。结果 优化工艺可获得具有纳米粒径的高收率外泌体（1.31 g/kg）,呈球形脂质双分子层结构,并对细胞无毒性。外泌体构建的纳米药物可被细胞摄取并分布于胞浆。外泌体负载基因可有效降低目的基因的表达水平（P <0.05）。结论 多步差速离心法和蔗糖密度梯度法可优化番茄外泌体的提取工艺,番茄外泌体可作为优良药物载体。

Optimization of tomato-derived exosomes isolation and analyzsisof feasibility of it as nano carriers

Objective To optimize extraction process of tomato-derived exosomes and preliminarilyexploration of the feasibility of tomato-derived exosomes as nano carriers. Methods Tomato-derived exosomes wereisolated by sucrose density gradient method. Dynamic light scattering and transmission electron microscopy wereused to identify the characteristics of exosomes, and MTT assay was used to detect the toxicity of exosomes in A549,A2780 and HONE1 (EBV+) cells. Tomato-derived exosomes were used to deliver model drug DiI and antisenseEBV-miR-BART7-3p to prepare nanomedicines. Their cellular uptake ability was tested in three cancer cells andthe silence effect of nano gene drugs was investigated by the fluorescent quantitative PCR. Results Exosomes withhigh yield (1.31 g/kg) were obtained by the optimized procedure. Tomato-derived exosomes exhibited nano-size andshowed the spherical lipid bilayer structure, and they had no significant cytotoxicity. The nanomedicine based onexosomes could be internalized in cells and distributed in the cytoplasm. The targeted gene expression level couldbe effectively silenced by gene drug, and the difference was statistically significant (P < 0.05). Conclusion Theextraction procedure of tomato-derived exosomes was optimized, and the feasibility of exosomes as excellent drugcarriers was verified.