Zhang Liping, Xu Yuan, Cao Wenjuan, Xie Shibao, Wen Lu, Chen Gang
School of Pharmacy.
Guangdong Provincial Key Laboratory of Advanced Drug Delivery.
Int J Nanomedicine. 2018 Jan 22;13:479-492. doi: 10.2147/IJN.S154968. eCollection 2018.
The round window membrane (RWM) functions as the primary biological barrier for therapeutic agents in the inner ear via local application. Previous studies on inner ear nano-drug delivery systems mostly focused on their pharmacokinetics and distribution in the inner ear, but seldom on the interaction with the RWM. Clarifying the transport mechanism of nanoparticulate carriers across RWM will shed more light on the optimum design of nano-drug delivery systems intended for meeting demands for their clinical application.
The poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) encapsulating coumarin-6 were prepared by emulsifying solvent evaporation method. We utilized confocal laser scanning microscope (CLSM) in combination with transmission electron microscope to investigate the transport pathway of PLGA NPs in the RWM. Simultaneously, the concentration and time dependence of NPs across the RWM were also determined. The endocytic mechanism of NPs through this membrane interface was classically analyzed by means of various endocytic inhibitors. The intracellular location of NPs into lysosomes was evaluated using CLSM scanning microscope colocalization analysis. The Golgi-related inhibitors were employed to probe into the function of Golgi and endoplasmic reticulum (ER) in the discharge of NPs out of cells.
PLGA NPs were herein transported through the RWM of a sandwich-like structure into the perilymph via the transcellular pathway. NPs were internalized predominantly via macropinocytosis and caveolin-mediated endocytic pathways. After being internalized, the endocytosed cargos were entrapped within the lysosomal compartments and/or the endoplasmic reticulum/Golgi apparatus which mediated the exocytotic release of NPs.
For the first time, we showed the translocation itinerary of NPs in RWM, providing a guideline for the rational fabrication of inner ear nanoparticulate carriers with better therapeutic effects.
圆窗膜（RWM）是内耳局部应用治疗药物的主要生物屏障。以往关于内耳纳米药物递送系统的研究大多集中在其药代动力学和在内耳中的分布，而很少关注与圆窗膜的相互作用。阐明纳米颗粒载体穿过圆窗膜的转运机制将为满足临床应用需求的纳米药物递送系统的优化设计提供更多启示。
采用乳化溶剂蒸发法制备包裹香豆素-6的聚乳酸-羟基乙酸共聚物纳米粒（PLGA NPs）。我们利用共聚焦激光扫描显微镜（CLSM）结合透射电子显微镜研究PLGA NPs在圆窗膜中的转运途径。同时，还测定了纳米粒穿过圆窗膜的浓度和时间依赖性。通过各种内吞抑制剂经典地分析纳米粒通过该膜界面的内吞机制。利用CLSM扫描显微镜共定位分析评估纳米粒在溶酶体中的细胞内定位。使用高尔基体相关抑制剂探讨高尔基体和内质网（ER）在纳米粒排出细胞中的作用。
PLGA NPs在此通过类似三明治结构的圆窗膜经跨细胞途径转运至外淋巴。纳米粒主要通过巨胞饮作用和小窝蛋白介导的内吞途径内化。内化后，内吞的货物被困在溶酶体区室和/或介导纳米粒胞吐释放的内质网/高尔基体中。
我们首次展示了纳米粒在圆窗膜中的转运行程，为合理制备具有更好治疗效果的内耳纳米颗粒载体提供了指导。
