Development of Protein Based PCBP2 siRNA Nanocomplex for Liver Fibrosis Therapy
Metadata[+] Show full item record
The objective of this dissertation is to develop a protein-based siRNA nanocomplex for the treatment of alcoholic liver fibrosis. Our laboratory recently discovered that silencing the poly (rC) binding protein 2 (PCBP2) gene in hepatic stellate cells (HSCs) leads to the reversal of the accumulated extracellular matrix. We therefore hypothesize that targeted delivery of the PCBP2 siRNA to HSCs could potentially treat liver fibrosis. Cholesterol and IGF2R (insulin growth factor 2 receptor) specific peptide were used as targeting ligands to deliver the siRNA to HSCs. In Chapter 1, we briefly introduced the background about RNA interference (RNAi), liver fibrogenesis, markers of liver fibrosis, and the role of PCBP2 in liver fibrogenesis. We also presented the Statement of the Problems and Objectives. In chapter 2, we reviewed the avidin-biotin technology and its potential applications in nanotechnology, therapy and diagnosis. We also discussed the challenges and biological barriers for siRNA delivery. In Chapter 3, we rigorously investigated the intracellular barrier which is a rate limiting step for the silencing activity of siRNAs. Using streptavidin as the nanocomplex core, PCBP2 siRNA was delivered to HSC-T6 (hepatic stellate) cells. Intracellular fate of the nanocomplex components and PCBP2 siRNA was monitored. Fluorescent probes were tagged with the siRNA, protamine and streptavidin and analyzed under confocal microscopy, flow cytometry and fluorescence spectroscopy. We discovered substantial exocytosis and localization of the siRNA in recycling organelles, such as recycling endosomes, endoplasmic reticulum and golgi apparatus. Streptavidin was found to be colocalized with the lysosomes and in some cases along with siRNA potentially leading to the lysosomal degradation. We found that the streptavidin, although, a very efficient delivery carrier has reduced silencing activity at higher incubation time intervals. In Chapter 4, we compared different variants of avidin such as avidin, neutravidin and streptavidin for in vitro activity and cellular uptake over the extended time interval. Addition of polyethylene glycol (PEG) spacer between the biotin and cholesterol ligand was done to improve the biodistribution of the nanocomplex. We tested the live imaging and post euthanized biodistribution of nanocomplexes and found it to be most distributed in liver in comparison to other variants of avidin. In vitro silencing activity and cellular uptake was also significantly higher in case of the neutravidin nanocomplex with negligible lysosomal colocalization and exocytosis. In Chapter 5, the neutravidin nanocomplex were further improved by using the IGF2R-specific peptide as a targeting ligand for hepatic stellate cells. The siRNA was also annealed to the peptide nucleic acid for attaching the biotin. PNA enhanced the serum stability of the siRNA and helped avoid the endonuclease and chemical reagent mediated degradation during biotin conjugation process. We developed the liver fibrosis model by injecting CCL4/olive oil intra-peritonealy for 4-5 weeks. We started the neutravidin-PCBP2 siRNA nanocomplex treatment at the beginning of 3rd week of the fibrosis induction to reverse the fibrosis. After the end of dosage regimen, the rats were euthanized and the analysis was performed for the liver fibrosis molecular markers. We found that the neutravidin PCBP2 siRNA nanocomplex successfully reversed the liver fibrosis by significantly reducing the molecular markers of fibrosis and reduction in the type 1 collagen.
Table of Contents
Introduction -- Literature review -- Inracellular fate and exocytosis of PCBP2 siRNA nanocomplex in hepatic stellate cells -- Conparison of avidin, neutravidin and streptavidin as nanocarriers for efficient siRNA delivery -- In vivo delivery of siRNA by protein based nanocomplex to treat aggressive liver fibrosis -- Summary and conclusions
Ph.D. (Doctor of Philosophy)