Faculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Japan.
Pharm Res. 2021 Mar;38(3):523-530. doi: 10.1007/s11095-021-03018-8. Epub 2021 Mar 15.
Food-derived nanoparticles exert cytoprotective effects on intestinal cells by delivering their cargo, which includes macromolecules such as microRNAs and proteins, as well as low-molecular weight compounds. We previously reported that apple-derived nanoparticles (APNPs) downregulate the expression of human intestinal transporter OATP2B1/SLCO2B1 mRNA. To verify the involvement of the cargo of APNPs in affecting the expression of transporters, we characterized the uptake mechanism of APNPs in intestinal cells.
The uptake of fluorescent PKH26-labeled APNPs (PKH-APNPs) into Caco-2, LS180, and HT-29MTX cells was evaluated by confocal microscopy and flow cytometry.
The uptake of PKH-APNPs was prevented in the presence of clathrin-dependent endocytosis inhibitors, chlorpromazine and Pitstop2. Furthermore, PKH-APNPs were incorporated by the HT29-MTX cells, despite the disturbance of the mucus layer. Additionally, the decrease in SLCO2B1 mRNA by APNPs was reversed by Pitstop 2 in Caco-2 cells, indicating that APNPs decrease SLCO2B1 by being incorporated via clathrin-dependent endocytosis.
We demonstrated that clathrin-dependent endocytosis was mainly involved in the uptake of APNPs by intestinal cells, and that the cargo in the APNPs downregulate the mRNA expression of SLCO2B1. Therefore, APNPs could be a useful tool to deliver large molecules such as microRNAs to intestinal cells.
食物衍生的纳米颗粒通过输送其货物（包括大分子如 microRNAs 和蛋白质以及低分子量化合物）对肠道细胞发挥细胞保护作用。我们之前报道过苹果衍生的纳米颗粒 (APNPs) 下调人肠转运蛋白 OATP2B1/SLCO2B1 mRNA 的表达。为了验证 APNPs 货物参与影响转运蛋白的表达，我们对 APNPs 在肠道细胞中的摄取机制进行了表征。
通过共聚焦显微镜和流式细胞术评估荧光 PKH26 标记的 APNPs（PKH-APNPs）在 Caco-2、LS180 和 HT-29MTX 细胞中的摄取。
在存在网格蛋白依赖性内吞作用抑制剂氯丙嗪和 Pitstop2 的情况下，PKH-APNPs 的摄取被阻止。此外，尽管干扰了粘液层，PKH-APNPs 仍被 HT29-MTX 细胞摄取。此外，APNPs 在 Caco-2 细胞中降低 SLCO2B1 mRNA 的作用被 Pitstop 2 逆转，表明 APNPs 通过网格蛋白依赖性内吞作用被内化从而降低 SLCO2B1 的表达。
我们证明了网格蛋白依赖性内吞作用主要参与了 APNPs 被肠道细胞摄取，并且 APNPs 中的货物下调了 SLCO2B1 的 mRNA 表达。因此，APNPs 可以成为将 microRNAs 等大分子递送至肠道细胞的有用工具。
