Title from PDF of title page viewed December 19, 2019Dissertation advisor: Kun ChengVitaIncludes bibliographical references (page 147-199)Thesis (Ph.D.)--School of Pharmacy and Department of Chemistry. University of Missouri--Kansas City, 2018The 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.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 conclusion
