Discovery of PSMA-Specific Peptide Ligands and Development of A Noncovalent Method to Attach Chemical Moieties to siRNA
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The objective of this dissertation is to discover PSMA (Prostate Specific Membrane Antigen)-specific peptide ligands using phage display and employ peptide nucleic acid as a complementary linker to attach chemical moieties to siRNA. In Chapters 1 and 2, we introduced and discussed the background of prostate cancer chemotherapy, PSMA specific ligands for tumor imaging and targeted drug delivery, mechanism and challenge of siRNA based therapeutics, as well as the advantages and clinical applications of peptide nucleic acids (PNAs). In Chapter 3, a PSMA specific peptide ligand was identified using phage display. A novel combinatorial phage biopanning strategy was employed to identify peptide ligands, which can specifically bind to PSMA. Among all candidate peptides, GTI peptide exhibits the highest affinity and the highest specificity to PSMA protein and PSMA positive cancer cells. When it is conjugated to pro-apoptotic KLA peptide, the GTI-KLA fusion peptide be can specifically uptaken by PSMA positive LNCaP cells and induce cell apoptosis. Biodistribution studies demonstrate that GTI peptide is specifically uptakes in PSMA positive tumors and this result indicates GTI peptide has great potential to be used as a ligand for targeted drug delivery for prostate cancer therapy. In Chapter 4, in order to improve the serum stability and increase the selectivity of siRNA, peptide nucleic acids (PNAs) were employed as a complementary bridge to link siRNA and targeting moieties. After optimizing the strategy, the siRNA-PNA chimera with a 9 mer PNA sequence annealed to the 5’ end of the antisense strand of siRNA exhibits the best serum stability and comparable silencing effect to free siRNA. This siRNA-PNA chimera platform was applied to deliver PCPB2 siRNA to high IGF2R expressed HSC-T6 cells by conjugation of a peptide-431, which is a targeting moiety of IGF2R. The formulated siPCPB2-PNA-peptide-431 demonstrates significantly higher cellular uptake and good silencing effect in HSC-T6 cells. In order to further increase silencing activity, biotin, instead of peptide-431, was used to conjugate PNA and formulate siPCPB2-PNA biotin, which was then mixed with biotin-peptide-431 and neutravidin to formulate neutravidin-based nanocomplex. This nanocomplex exhibits excellent silencing activity in vitro.
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Introduction -- Literature review -- Discovery of PSMA-specific peptide ligands for targeted drug delivery -- Development of noncovalent method to attach chemical moieties to siRNA -- Summary and conclusions
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Ph.D.