Intracellular trafficking and exocytosis of a multi-component siRNA nanocomplex

Abstract

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 form 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.

Targeted Delivery of an siRNA/PNA Hybrid Nanocomplex Reverses Carbon Tetrachloride‐Induced Liver Fibrosis

“…In the end of the study, the rats were euthanized, and the serum and liver were harvested. [16] Herein, we also observed downregulation of type I collagen mRNA in the liver after treatment with the PCBP2 siRNA nanocomplex ( Figure 6D). In comparison to healthy rats, CCl 4 induction dramatically increased ALT and AST values to 500 IU L −1 and 464 IU L −1 , respectively, indicating significant inflammation and damage in the liver.…”

“…We previously demonstrated that silencing PCBP2 in HSCs reduced the half-life of type I collagen mRNA and subsequently downregulated the amount of the mRNA. [16] Herein, we also observed downregulation of type I collagen mRNA in the liver after treatment with the PCBP2 siRNA nanocomplex ( Figure 6D). This downregulation is the key for the regression of accumulated ECM in fibrotic liver.…”

“…We therefore aimed to increase the molar ratio of siRNA in the nanocomplex to increase drug loading (Figure 2), thus minimizing potential side effects associated with the delivery system. 2019, 2 There are several molecular receptor that have been investigated for HSC-targeted delivery, such as low density lipoprotein receptor (LDLR), [16,23] cellular retinol binding receptor protein 1 (CRBP-1), [24] and IGF2R. Meanwhile, the siRNA nanocomplex with the optimized molar ratio shows a very small particle size (ß148 nm), which will be helpful in crossing the liver sinusoids ( Figure 2).…”

Cholesterol-Containing Nuclease-Resistant siRNA Accumulates in Tumors in a Carrier-free Mode and Silences MDR1 Gene

“…The issue of toxicity is also very important 41 . Phagocytic uptake by the mononuclear phagocyte system in the bloodstream is a major challenge when using nanoparticles for siRNA delivery, which forces the use of chemical shields (for example, polyethylene glycol [PEG]) to prevent recognition 42 .…”

The principles and applications of avidin-based nanoparticles in drug delivery and diagnosis

“…As shown in Figure 2B, the biotinylated siRNA and biotinylated cholesterol anchored to the streptavidin backbone provide efficient silencing of the PCBP2 gene for liver fibrosis treatment. [57, 58] Streptavidin-biotin technology has also been utilized for the delivery of Kv1.3 siRNAs in the treatment of autoimmune disorders. Biotinylated polyethylene glycol and cholesterol were functionalized with biotinylated-CD45RO antibodies through the use of streptavidin to provide highly specific targeting and uptake of nanoparticles by Memory T cells, which had the consequence of decreasing the calcium ion influx, thus producing a therapeutic effect.…”