Intracellular trafficking and exocytosis of a multi-component siRNA nanocomplex

Despite the importance of siRNA delivery systems, understanding of their intracellular fate remains elusive. We recently developed a multi-component siRNA nanocomplex to deliver siRNA to hepatic stellate cells (HSCs). The objective of this study is to study post-internalization trafficking of this siRNA nanocomplex and its multiple components like siRNA, protamine, and streptavidin, in HSCs. After internalization, the nanocomplex entrapped in early endosomes undergoes three possible routes including endosomal escape, exocytosis, and entrapment in lysosomes. Significant amount of siRNA dissociates from the nanocomplex to exert silencing activity. After escaping from endosomes, protamine dissociates from the nanocomplex and stays inside the cytoplasm. Golgi complex plays an important role in exocytosis of the nanocomplex. We also demonstrate that exocytosis is one of the major reasons accounting for the transient silencing activity of nonviral siRNA delivery. Incorporation of exocytosis inhibitors in nonviral siRNA delivery systems may extend the silencing activity of siRNA.

Although endocytosis of nanocarriers has been extensively studied, intracellular trafficking and exocytosis of nanocarriers have always been elusive and ignored. Previously, we developed a multi-component streptavidin-based siRNA nanocomplex to deliver the PCBP2 siRNA into rat hepatic stellate cells (HSCs). The study aims to utilize confocal microscopy and flow cytometry to precisely study the intracellular trafficking of each of the major components, including the siRNA, protamine, and streptavidin of the nanocomplex. Our studies particularly indicate the importance of exocytosis in nonviral siRNA delivery systems. We demonstrate that exocytosis is one of the major reasons for the transient silencing activity of nonviral siRNA delivery. Therefore, further study and understanding of the exocytosis of nanocarriers may provide new insights for improving nanoscale drug delivery systems.Figure optionsDownload high-quality image (190 K)Download as PowerPoint slide