Peptide-Based Platform For Cancer Therapeutics And Drug Transportation Across Blood-Brain Barrier
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Prostate cancer is the most common male malignancy and remains the second-leading cause of death due to cancer in American men. While surgery and radiation are effective in prostate cancer therapy at early stages, the majority of the patients ultimately relapse and develop advanced prostate cancer. Conventional chemotherapy is one of the most widely used techniques to combat prostate cancer, along with surgery, radiation therapy, and cryotherapy. However, chemotherapy itself has many limitations, such as lack of specificity, high toxicity to normal cells, inadequate availability to the tumor tissue, drug resistance, and the complicated microenvironment in tumor tissues. As a result, there is a great need to develop targeted delivery systems for chemotherapy agents for prostate cancer therapy. One objective of this dissertation is to develop peptide-based platforms for two chemotherapy agents, TGX-D1 and cabazitaxel. Despite efforts and advancements in the field of neuroscience, drug delivery to the brain and CNS remains a major challenge. Neurological diseases/disorders, infections, and brain cancers are becoming more predominant but effective drug therapies remain elusive. The blood-brain barrier (BBB) is a physical, chemical and metabolic barrier that restricts and controls the movement of substances from the blood circulation to the brain extracellular fluid or brain cells. Because of this barrier, approximately 98% of small-molecule drugs and 100% of large-molecule drugs, including recombinant proteins, monoclonal antibodies, and nucleic acids, cannot cross the BBB. The second objective of this dissertation is to identify a peptide sequence that can cross the BBB and deliver drug molecules, without affecting the integrity of the BBB. This work will be completed using the phage display technique. In chapter 1 and 2, we introduced and discussed the background of prostate cancer and the utilization of prostate cancer relevant antigens and enzyme for targeted drug delivery. We also discussed the role of the BBB as a major hurdle in drug delivery to the brain, and the identification of peptide ligands using phage display. In chapter 3, an enzyme-responsive peptide drug conjugate was developed for TGX-D1, a promising PI3K inhibitor for prostate cancer therapy. LNCaP-specific KYL peptide was used as the targeting ligand and the prostate-specific antigen (PSA) cleavable peptide (SSKYQSL) was used as the enzyme-responsive linker. SSKYQSL is cleaved by recombinant human PSA at 10-250 μg/mL. By contrast, the linker is stable in the serum of prostate cancer patients with high PSA levels (> 500 ng/mL), indicating that this linker can survive the systemic circulation in prostate cancer patients but be cleaved in the tumor microenvironment. Cellular uptake of the peptide drug conjugate in prostate cancer cells is improved by about nine times. Biodistribution studies reveal significant tumor accumulation of the peptide drug conjugate in nude mice bearing C4-2 tumor xenografts. Meanwhile, distribution of the conjugate in other major tissues is the same as the parent drug, indicating a high specificity of the conjugate to prostate cancers in vivo. In chapter 4, we developed a biodegradable, enzyme-responsive, and targeted polymer micelle for cabazitaxel. The micelle was spontaneously formed from two amphiphilic block copolymers. The first block copolymer consists of PEG, an enzyme-responsive peptide, and cholesterol; whereas the second block copolymer consists of a targeting ligand (DUPA), PEG and cholesterol. The enzyme-responsive peptide is cleavable in the presence of MMP-2, which is overexpressed in prostate cancer cells. DUPA is a small molecule-based targeting ligand, which binds to prostate-specific membrane antigen (PSMA) with a high affinity. The micelle showed a low critical micelle concentration (CMC), high drug loading, and high entrapment efficiency. The release of cabazitaxel from the micelle is dependent on the cleavage of the enzyme-responsive peptide by MMP-2. Also, the micelle showed dramatically higher cellular uptake in prostate cancer cells compared to free cabazitaxel. Importantly, the polymeric micelle demonstrated better inhibition of tumor growth in mice bearing prostate cancer xenografts compared to free cabazitaxel. Taken together, these findings suggest that the enzyme-responsive PSMA-targeted cabazitaxel micelle is a potent and promising drug delivery system for advanced prostate cancer therapy. In chapter 5, we developed a novel phage display biopanning procedure to discover shuttle peptides that can cross the BBB. 7-mer and 12-mer peptide phage libraries were used in the study. For the 7-mer phage library, we used a five-round biopanning procedure consisting of two rounds of in vitro screening and three rounds of in vivo screening. We selected more than 100 phage colonies for DNA extraction and sequencing. We successfully discovered 62 novel peptide sequences, of which seven sequences repeated multiple times for a total repetition of 50 times. These sequences were used for in vivo biodistribution studies. We labeled these peptides with a fluorescent dye and injected them into rats via the tail vein. We then euthanized the rats, collected various organs, and checked the fluorescence intensity in each of the organs. For the 12-mer library, we designed a 4-round biopanning procedure. We started with a first round of in vitro selection, followed by in vivo screening, and then repetition of the in vitro and in vivo rounds of screening. We dissected different parts of the brain, including the cerebellum, brain stem, and cerebrum, and enriched phages separately for each part of the brain to enrich peptides that are specific for particular parts of the brain. We successfully discovered more than 150 novel peptide sequences, with more than 50 sequences specific for different parts of the brain
Table of Contents
Introduction -- Literature review -- An Enzyme-Responsive Conjugate Improves the Delivery of a P13K Inhibitor to Prostrate Cancer -- Cabazitaxel-loaded Enzyme-Responsive Polymeric Micelle for Targeted Prostrate Cancer Therapy -- Discovery of Blood-brain Barrier Shuttle Peptide Using Phage Display Technique -- Appendix
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Ph.D. (Doctor of Philosophy)