[-] Show simple item record

dc.contributor.advisorCheng, Kun (Professor)
dc.contributor.authorChen, Zhijin
dc.date.issued2016
dc.date.submitted2016 Fall
dc.descriptionTitle from PDF of title page, viewed January 3, 2018
dc.descriptionDissertation advisor: Kun Cheng
dc.descriptionVita
dc.descriptionIncludes bibliographical references (pages 99-109)
dc.descriptionThesis (Ph.D.)--School of Pharmacy, Department of Chemistry and School of Medicine. University of Missouri--Kansas City, 2016
dc.description.abstractThe objective of this dissertation is to present two approaches to identify Hepatic Stellate Cells (HSCs) targeting ligands using phage-display library biopanning and systematic evolution of ligands by exponential enrichment (SELEX). These HSC-specific ligands can be used for diagnosis and therapy of liver fibrosis. The mechanism of fibrogenesis and potential treatments of liver fibrosis are summarized in Chapters 1 and 2. In Chapter 3, we identified HSC-specific peptides using a peptide phage display for diagnosis and treatment of liver fibrosis. Caused by chronic injuries, such as hepatitis, alcohol abuse, and nonalcoholic steatohepatitis, liver fibrosis is characterized by the excessive accumulation of extracellular matrix (ECM) in the liver. Activation of HSCs is the most critical step during liver fibrogenesis due to the production of excessive ECM and profibrogenic cytokines. Therefore, development of HSC-specific delivery systems is essential for the success of antifibrotic agents. The objective of this Chapter is to identify peptide ligands targeting the insulin like growth factor II receptor (IGFIIR), which is overexpressed in HSCs during liver fibrogenesis. A combinatorial phage display biopanning against IGFIIE protein- and HSC-T6 rat hepatic stellate cells was conducted to identify phage/peptide candidates. Phage ELISA, cellular uptake and cell viability assay were employed to evaluate the binding affinity and specificity of these peptide ligands to recombinant human IGFIIR and HSCs. IGFIIR siRNA was used to silence the IGFIIR expression in human HSCs (LX-2) to confirm the specificity of the identified peptide ligands. Among the identified peptide candidates, the peptide-431 shows the highest binding affinity and specificity to recombinant human IGFIIR protein and HSCs. The apparent equilibrium dissociation constant (Kd) of the peptide-431 is 6.19 µM for LX-2 cells and 12.35 μM for rat hepatic stellate cells HSC-T6. Cellular uptake of the peptide-431 in LX-2 cells is significantly reduced after silencing IGFIIR with siRNA. The peptide-431 also enhances the uptake of a proapoptotic peptide (KLA peptide) in LX-2 and HSC-T6 cells, indicating that the peptide-431 can be used as a targeting ligand to deliver antifibrotic agents into not only rat but also human HSCs. In order to improve the potential of the application of peptide-431 in liver fibrosis targeted delivery, we developed a dimerized peptide-431 which is also descripted in Chapter 3. The binding affinity of peptide-431 has improved 9 fold by forming dimer comparing with monomer peptide-431. We also screened aptamer ligands using SELEX for targeted delivery to HSCs. In Chapter 4, IGFIIR-specific aptamers were identified using SELEX. The binding affinity and specificity of the aptamers were studied by flow cytometry and Surface Plasma Resonance (SPR). The identified aptamer was annealed with the PCBP2 siRNA and delivered it into HSCs. The recovered aptamers result shows that the aptamers were enriched after seven rounds of SELEX. The binding affinity (Kd) of the aptamer-20 on IGFIIR protein is approximately 35.5 nM. Knocking down the expression of IGFIIR with siRNA decreased the binding affinity of aptamer-20, indicating the specificity of the aptamer to IGFIIR in HSCs. Cellular uptake of the aptamer-siRNA chimera is much higher than the siRNA. PCBP2 expression in HSCs is significantly downregulated after incubation with the aptamer-siRNA chimera in LX-2 cells. After systemic administration, the aptamer-siRNA chimera is primarily located in the liver of rats with CCl₄-induced liver fibrosis. Therefore, aptamer-20 is a very promising IGFIIR-specific ligand for drug delivery to HSCs.eng
dc.description.tableofcontentsIntroduction -- review of literature -- Discovery of peptide ligands for hepatic stellate cells using page display -- Discovery of aptamer ligands for hepatic stellate cells using SELEX -- Summary and conclusion -- Appendix letter of permission
dc.format.extentxiii, 114 pages
dc.identifier.urihttps://hdl.handle.net/10355/62413
dc.publisherUniversity of Missouri--Kansas Cityeng
dc.subject.lcshDrug delivery systems
dc.subject.lcshLiver cells
dc.subject.meshDrug Delivery Systems
dc.subject.meshHepatocytes
dc.subject.otherDissertation -- University of Missouri--Kansas City -- Pharmacy
dc.subject.otherDissertation -- University of Missouri--Kansas City -- Chemistry
dc.subject.otherDissertation -- University of Missouri--Kansas City -- Medicine
dc.titleDiscovery of Peptide and Aptamer Ligands for Targeted Drug Delivery to Hepatic Stellate Cellseng
dc.typeThesiseng
thesis.degree.disciplinePharmaceutical Sciences (UMKC)
thesis.degree.disciplineChemistry (UMKC)
thesis.degree.disciplineBiomedical and Health Informatics (UMKC)
thesis.degree.grantorUniversity of Missouri--Kansas City
thesis.degree.levelDoctoral
thesis.degree.namePh.D.


Files in this item

[PDF]

This item appears in the following Collection(s)

[-] Show simple item record