Pharmaceutical Sciences Electronic Theses and Dissertations (UMKC)
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The items in this collection are the theses and dissertations written by students of the Division of Pharmaceutical Sciences. Some items may be viewed only by members of the University of Missouri System and/or University of Missouri-Kansas City. Click on one of the browse buttons above for a complete listing of the works.
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Item An electrochemical pipette with thin-layer design for rapid electrosynthesis and mechanistic study of the catalytic reactions(2025) Punchihewa, Buwanila Tharusha; Rafiee, Mohammad; Gutheil, WilliamIn the past few decades, electrochemical organic synthesis has emerged as a transformative approach to modern organic synthesis. Electrochemical methods enable unique reaction pathways for both direct (electrode-driven) and mediated (redox-catalyst-assisted) systems through controlled electron transfer, access to reactive intermediates, and transformations that are inaccessible to conventional methods. However, traditional electrochemical reaction setups depend on mass transport, and extended electrolysis times limit their application in organic synthesis. Therefore, developing an electrochemical design that is independent of mass transfer, and facilitating fast electrolysis is essential for advancing the field of electrochemical organic synthesis. In this study, we fabricated an electrochemical cell that confines the electrolysis volume to a thin layer of solution, comparable to the thickness of the diffusion layer. The fabricated thin layer electrode (TLE) is independent of mass transfer and allows electrolysis to be performed within minutes owing to its high surface (A)/ volume (v) ratio. The utility of the TLE electrode for parallel electrosynthesis applications was benchmarked using N-hydroxyphthalimide (NHPI) mediated electrochemical C-H functionalization. Rapid electrolysis and generation of microscale volumes make TLE suitable for application in drug metabolism. The application of TLE in drug metabolism has been demonstrated through the oxidative metabolism of acetaminophen under mild basic and acidic conditions. The formation of an N-acetyl-p-benzoquinone imine metabolite (NAPQI) during acetaminophen oxidation and its subsequent chemical reactions during electrolysis were successfully studied. Moreover, the integration of a microelectrode with TLE (combined TLE) enabled the real-time monitoring of redox-active intermediates formed during rapid electrosynthesis. Real-time studies of electrocatalytic cycles allow for a better understanding of electrocatalytic mechanisms and their resting states. The utility of this combined TLE for mechanistic studies of electrochemical synthesis was demonstrated using a Ni-catalyzed biaryl coupling reaction. This approach allowed the real-time monitoring of both closed- and open-shell nickel species generated during nickel-catalyzed biaryl coupling reactions under catalytic conditions. Studying both the closed- and open-shell nickel species generated during the catalysis cycle using spectroscopic methods is challenging. Therefore, this approach assists in understanding the unreported trends in Ni speciation, concentration profiles of key intermediates under different reaction conditions, and reaction progress.Item Delivery of sting agonist using lipid nanoparticles and discovery of anti-TIGIT peptides for cancer therapy(2023) Shaji, Sherin George; Cheng, Kun (Professor); Yao, XiaolanOver the past decades, cancer treatment has significantly advanced with the approval of new therapeutic entities aimed at treating this disease. However, these advancements have not universally benefitted all patients or different cancers due to the complexity of the tumor microenvironment found in these malignancies. Therefore, it has become crucial to understand the molecular and cellular mechanisms that cancer cells use to evade the immune system of their hosts in order to discover effective therapeutic interventions. Tumors can be classified based on their immunogenicity into two types: cold and hot tumors. Cold tumors like pancreatic cancer, glioblastoma, prostate cancer, and ovarian cancer exhibit a high number of immune suppressive cells including regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSC), and cancer-associated fibroblasts (CAFs). Additionally, they often lack significant infiltration of immune cells like T cells. On the other hand, hot tumors contain T cells and other immune cells within the tumor microenvironment. However, despite the presence of these immune cells, cancer cells utilize various mechanisms to render the immune cells, especially T cells, dysfunctional, thereby evading the immune system. One significant mechanism involves immune checkpoints, where cancer cells manipulate the interaction between a receptor on T cells and a ligand on cancer cells. This manipulation often involves the cancer cells overexpressing their specific ligands, leading to a phenomenon where T cells become exhausted and unable to effectively eliminate tumor cells. Cold tumors can be treated by converting them into hot tumors and then removing the brakes by inhibiting the receptor-ligand interaction. This dissertation focuses on two main research objectives related to the interventions mentioned above. The first research aims to deliver a STING agonist into a cold tumor using lipid nanoparticles. This delivery is intended to provoke an immune response that leads to the elimination of tumor cells. STING pathway agonism has shown promise in innate immune signaling to tune the tumor microenvironment toward an immunogenic phenotype by promoting immune cell infiltration, especially CD8+ T cells, in various types of cancer. The second research involves the discovery of anti-TIGIT cyclic peptides using phage biopanning to block the interaction between the receptor TIGIT and its high-affinity ligand, CD155. In clinical studies, inhibiting this pathway within the adaptive immune system has demonstrated the ability to reverse the exhaustion of T- and NK cells, thereby restoring their functional ability to elicit cytotoxic activity against tumor cells. Chapter 1 introduces the dissertation research, presenting the statement of problems and outlining the research objectives. Chapter 2 provides an in-depth literature review on pancreatic cancer, the STING-2’3’-cGAMP pathway activation, and TIGIT-CD155 checkpoint inhibition for cancer immunotherapy. Chapter 3 involves the development and characterization of 2’3 cGAMP lipid nanoparticles (cGAMP-LNP) using various in-vitro and in-vivo techniques. These biodegradable cGAMP-LNP were prepared by encapsulating 2’3' -cGAMP within a lipid-based system containing an ionizable lipid, LHHK, and co-lipids. The cGAMP-LNP were found to have higher cellular uptake, improved IRF activation, and endosomolytic activity compared to free cGAMP in various cell-based assays. The cGAMP-LNP demonstrated significant inhibition of pancreatic cancer growth in a mouse model. Furthermore, these LNPs displayed a good safety profile in both in-vitro and in-vivo testing. These results suggest that cGAMP-LNP is a promising therapeutic entity for cold tumors like pancreatic cancer. In Chapter 4, the dissertation illustrates the discovery of anti-TIGIT cyclic peptides using the phage display technique. A unique cyclic peptide library developed in our laboratory was utilized to identify cyclic peptides using a solution-based biopanning procedure. These peptides were selected for their ability to bind to the TIGIT protein and block the TIGIT-CD155 interaction. The blocking efficiency was analyzed using an in-vitro protein-based assay, and two peptides, CSCP-7, and CSCP-16, showed the highest blocking of TIGIT-CD155 interaction. Moreover, alanine scanning revealed the amino acids responsible for the blocking activity of the CSCP-16 peptide.Item Exploring multivalency and chemical modifications in the development of anti-PD-L1 peptide inhibitors for cancer immunotherapy(2023) Mamani, Umar-Farouk Wumbei; Cheng, Kun (Professor); Peng, Zhonghua, Ph. D.The PD-1/PD-L1 immune checkpoint axis is one of the most common pathways exploited by cancers to evade immune surveillance. Many cancers overexpress PD-L1 (programmed cell death ligand 1) on their surface through which they interact with PD-1 (programmed cell death protein 1) on activated immune cells, leading to suppression of the immune response. Significant efforts have been made in oncology research to block the PD-1/PD-L1 interaction. The most common approach is using monoclonal antibodies (mAbs) to competitively antagonize the PD-1/PD-L1 interaction. Among other qualities, the multivalent nature of antibodies (Abs) gives them a high affinity for targets and makes them attractive agents for blocking the PD-1/PD-L1 interaction. However, despite some successes achieved with anti-PD-L1/anti-PD-1 mAbs, there are growing number of reports of suboptimal responses. Additionally, many patients develop resistance and experience immune-related adverse effects due to drawbacks associated with Abs, such as low tumor penetration, high immunogenicity, and prolonged occupancy of target receptors. Over the past decade, peptides have garnered considerable interest as promising alternatives to address the limitations of existing anti-PD-L1 therapies and to overcome some of the drawbacks of mAb-based treatments. Compared to mAbs, peptides have better cell permeability, cost-effectiveness, and the ability for easy formulation for non-parenteral administration. However, the successful translation of anti-PD-L1 peptides is often hindered by challenges related to their limited stability and efficacy. Peptides are typically monovalent and exhibit lower affinities for targets compared to mAbs, which are multivalent. Our group recently discovered a 9-amino acid anti-PD-L1 peptide, TR3. Despite its reasonable potency, it still exhibits poor stability. In this study, we aim to mimic the multivalent nature of antibodies by designing multivalent anti-PD-L1 peptides and exploring how valency influences the blocking efficacy, potency, stability, anti-cancer effect, and tumor penetration. We also aim to optimize the sequence and structure of TR3 to develop more potent and stable TR3 derivatives. This involves modifying sidechains and backbones of the peptide. In the first chapter, we provide an overview of the dissertation research, highlighting checkpoint blockade-based cancer immunotherapy and the significance and challenges of peptides-based therapeutics as potential checkpoint blockade agents. We also present the problems this dissertation aims to address and discusses its objectives. Chapter 2 thoroughly explores the development of peptides as therapeutic agents, focusing on their pivotal role in cancer therapy. We further discuss cancer immunotherapy and the different modalities, especially checkpoint blockade therapy. The chapter concludes with an in-depth examination of the PD-1/PD-L1 pathway and critically reviews the different strategies used to block this interaction. In Chapter 3, we detail our efforts in developing multivalent anti-PD-L1 peptide inhibitors for cancer immunotherapy. We demonstrate that increasing the peptide valency improves its blocking efficiency, with the tetrameric peptide showing a 39-fold improvement over the original peptide. The inhibitory potential of the multivalent peptides was also demonstrated in co-culture experiments involving human peripheral blood mononuclear cells (PBMCs) and DU145 cancer cells. These peptides significantly suppressed cancer cell-mediated T-cell apoptosis, revitalizing T-cells to inhibit the proliferation of cancer cells. Notably, while increasing the valency improved the peptide’s activity, the magnitude of the increase tended to diminish with each subsequent increase in valency. Also, increasing the valency did not adversely affect the peptide’s stability, and the multivalent peptides exhibited similar stability profiles as the monomeric peptide. Peptides achieve good tumor penetration due to their relatively small sizes. A primary concern regarding multimerization of ligands is the corresponding size increase. Using a 3D tumor spheroid model, we showed that the multivalent TR3 peptides had comparable tumor penetration as the monomeric TR3. Additionally, TR3 and TR3-tetramer (TR3-T) exhibited similar tumor distribution profiles in biodistribution studies. However, the multivalent TR3 peptides had significantly higher binding to human serum albumin than the monomeric TR3. While this may be beneficial for achieving a prolonged anti-tumor effect, it could potentially reduce the amount of unbound peptide available for activity. This characteristic necessitates additional consideration when designing multivalent anti-PD-L1 inhibitors. Chapter 4 details our research in developing stable anti-PD-L1 peptides for cancer immunotherapy. In this study, we employed various peptide optimization strategies such as alanine scanning, sidechain optimization, and substitutions with D- and N-methyl amino acids to design peptidomimetic analogs of TR3. These modifications improved the stability of the resulting analogs by about 5-fold. The analogs also exhibited enhanced anti-PD-L1 blocking potency with IC₅₀ values of 355 nM, 359 nM, and 165 nM for TR3-29, TR3-30, and TR3-64, respectively, compared to TR3 with an IC₅₀ of 5,404 nM. The improved activity of the analogs was also evident in an in-vitro coculture experiment involving human PBMCs and DU145 cancer cells. The TR3 analogs showed a significantly higher effect in reducing T-cell apoptosis and inhibiting the proliferation of DU145 cells compared to TR3. Among the analogs, TR3-64 showed the best effect and was further multimerized into dimeric and tetrameric TR3-64 analogs (i.e., TR3-64D and TR3-64T respectively) with IC₅₀ values of 57 nM and 22 nM, respectively. TR3-64 also showed a significant effect in suppressing the growth of murine CT-26 colorectal tumors in a xenograft model. Despite their improved serum stability, the TR3-analogs still exhibited poor gastrointestinal stability. Hence, we used a D-amino acid scan and enzyme-targeted D-amino acid modifications to improve the stability of the peptides. We identified two candidates: TR3-114 and TR3-115, with good serum and gastrointestinal stability and blocking efficiency. TR3-114 and TR3-115 had blocking potency IC₅₀ values of 172 nM and 665 nM, respectively. The peptides also showed good serum stability with half-lives exceeding 24 hours.Item Discovery of IKBKE and CD24 siRNAs for the treatment of triple-negative breast cancer(2023) Liu, Yanli, 1988-; Cheng, Kun (Professor); Mohan, Ryan D.Triple-negative breast cancer (TNBC) represents approximately 10-20% of all newly diagnosed breast cancers and is classified as a subtype with the absence of ER, PR, and HER2 expression. TNBC displays high aggressiveness, a tendency to metastasize, a poor prognosis, and a low survival rate. Unlike other breast cancer subtypes, TNBC has limited treatment options due to the lack of targeted therapies. Therefore, there is a significant need to develop new therapies for TNBC. IKBKE, also known as IKKε or IKKi, is a member of the IKK (IκB kinase) family and exhibits high expression levels in various cancers. IKBKE functions as an oncogene in breast cancer and is overexpressed in approximately 30% of breast carcinomas. IKBKE is shown to be aberrantly amplified in TNBC and associated with proliferation, migration, and survival in TNBC cells. Breast cancer also expresses high levels of CD24, which is a heavily glycosylated glycosylphosphatidylinositol (GPI)-anchored surface protein and plays an important role in tumor growth, invasion, and metastasis. Moreover, overexpression of CD24 in tumors is associated with resistance to therapies. Small interfering RNA (siRNA) can specifically knockdown the expression of target genes. It represents a promising tool for cancer therapy as it can silence aberrant genes that are essential for the progression of cancer cells. However, successful siRNA cancer therapy relies on the development of safe and effective RNA delivery systems because naked siRNA is unstable and has limited cellular uptake. Numerous delivery carriers have been investigated to increase the stability and improve the cellular uptake of siRNAs. This dissertation focuses on two primary research objectives. The first objective centered on discovering siRNAs targeting IKBKE and CD24 for the treatment of TNBC. The second research objective aims to utilize a recently discovered anti-PD-L1 human domain antibody as an immune checkpoint inhibitor for cancer immunotherapy. In Chapter 1, we briefly introduced the background of the research, the statement of the problems, and research objectives. In Chapter 2, we reviewed potential treatment options for TNBC and provided a concise introduction to cancer immunotherapy, specifically focusing on PD-1/PD-L1 immune checkpoint inhibitors. In Chapter 3, we utilized a cholesterol peptide-based delivery system to condense IKBKE siRNA to form nanocomplexes for the treatment of TNBC. The stability, cellular uptake, and penetration capability of the cholesterol peptide/siRNA (CCP/siRNA) nanocomplex was significantly increased. Importantly, the CCP/siRNA nanocomplex significantly inhibited tumor growth in an orthotopic TNBC mouse model. These data suggest that IKBKE siRNA could be a promising therapeutic strategy for TNBC. In Chapter 4, we designed four CD24 siRNAs for the treatment of TNBC by targeting different regions of human CD24 mRNA. The results showed the pre-designed siRNA reduced the CD24 expression at both mRNA and protein levels in TNBC cells. CD24 siRNA efficiently inhibited the proliferation, migration, and invasion of TNBC cells. Moreover, silencing of CD24 induced tumor cell apoptosis and cell cycle arrest. Further, we evaluated the association of CD24 expression with doxorubicin resistance. We found that both CD24 mRNA and protein levels were upregulated in doxorubicin-treated MDA-MB-231 cells and CD24 siRNA sensitized MDA-MB-231 cells to doxorubicin which was reflected by a decreased IC50 value. Overall, targeting CD24 with siRNAs may be a promising therapeutic target for TNBC or other cancers with overexpressed CD24. In Chapter 5, we presented the work on the discovery of anti-PD-L1 human domain antibodies (dAbs) using the phage display technique for cancer immunotherapy. In this study, seven anti-PD-L1 domain antibodies were discovered. Among them, the CLV3 dAb showed the highest binding affinity to human and mouse PD-L1 proteins with KD values of 137.5 nM and 266.8 nM, respectively. The CLV3 dAb also exhibited potent binding affinity to PD-L1 overexpressing DU145 cells. CLV3 inhibited PBMC apoptosis in a co-culture system and showed better tumor penetration compared to antibody. The CLV3 dAb significantly inhibited tumor growth and increased the survival of CT26 tumor-bearing mice. The CLV3 dAb is therefore a promising PD-L1 inhibitor that can be used for cancer immunotherapy.Item Targeting extracellular matrix using siRNA for the treatment of liver fibrosis and pancreatic cancer(2023) Lin, Chien-Yu; Cheng, Kun (Professor)The objective of this dissertation is to develop peptide-based siRNA delivery systems for the treatment of liver fibrosis and pancreatic ductal adenocarcinoma (PDAC). We recently discovered that silencing the poly (rC) binding protein 2 (PCBP2) gene in hepatic stellate cells (HSCs) and NIH 3T3 fibroblasts can reverse the accumulation of extracellular matrix in liver fibrosis and improve the penetration of small molecules in the tumor microenvironment, respectively. We previously developed a small peptide-based delivery system primarily composed of lysine and histidine residues, which improved the bioactivity and stability of siRNA. We, therefore, hypothesize that cholesteryl peptide/PCBP2 siRNA nanocomplexes modified with targeting and functional polyaminoacids/polypeptides could increase the specificity, delivery efficiency, and therapeutic efficacy. Furthermore, we also developed and evaluated a feasible rat model for alcoholic liver fibrosis. In Chapter 1, we briefly introduced the background of the dissertation research and presented the Statement of the Problems and Objectives. In Chapter 2, we reviewed the liver fibrogenesis and potential treatments for liver fibrosis. We also discussed the strategies to target desmoplastic stroma and cancer immunotherapy for pancreatic cancer. In Chapter 3, we presented the development of peptide-based siRNA nanocomplexes for HSC-specific drug delivery. We recently discovered a 12-mer ligand peptide for insulin-like growth factor 2 receptor (IGF2R) using the phage display technique, which significantly increased the binding affinity of nanoparticles to activated HSCs. In this chapter, we employed the IGF2R ligand peptide to improve the silencing efficiency and cellular uptake of the cholesteryl peptide/PCBP2 siRNA nanocomplexes. We incorporated glycine spacer and glutamate residues into the C-terminus of the dimeric IGF2R peptide ligands to non-covalently modify the surface of the siRNA nanocomplexes. We then compared the in vitro features of various IGF2R peptide-modified nanocomplexes. The resulting nanocomplexes demonstrated improved silencing efficiency activity, cellular uptake, and accumulation in the liver of rats with CCl₄-induced liver fibrosis. This strategy provides a promising platform for delivering antifibrotic siRNAs for liver fibrosis. In Chapter 4, we presented the development of a feasible rat model for alcoholic liver fibrosis and discussed its key characteristics. To replicate human drinking patterns, the rats were fed with a daily alcohol liquid diet and multiple alcohol binges, mimicking chronic and acute alcohol intake in humans, respectively. Carbon tetrachloride (CCl₄), the most commonly used hepatotoxin to induce liver fibrosis and cirrhosis in rodents, was administrated at a low dose to accelerate liver fibrogenesis. We discussed the pathophysiological features of liver injury and fibrosis induced by the combination of alcohol and low-dose CCl₄ treatment with various patterns. The results showed that the combination of alcohol and low-dose CCl₄ had a synergistic effect on increasing the levels of liver enzyme activity, collagen expression, HSCs activation, and cell proliferation. Consequently, this led to the progression of liver fibrosis. We successfully established an 8-week rat model of alcoholic liver fibrosis, providing a time-efficient model for future studies. In Chapter 5, we described a novel combination therapy approach for pancreatic ductal adenocarcinoma (PDAC). We developed a nanocarrier for delivering PCBP2 siRNA and an anti- programmed cell death ligand-1 (PD-L1) peptide simultaneously, aiming to improve the antitumor efficacy and delivery efficiency. Phosphorothioate (PS) linkage and 2’-O-methyl (2’-OMe) modified PCBP2 siRNA showed a longer half-life in mouse serum compared with unmodified PCBP2 siRNA. To increase the accumulation of siRNA and anti-PD-L1 in the tumor, the cholesteryl peptide/siRNA nanocomplexes was further condensed with plectin-1-G-pE6-M-PD-L1 (PGEMP) polypeptide. We assessed the characteristics of the resulting nanocomplexes, including particle size, cell viability, silencing activity, and serum stability. This strategy provides a promising platform for improving the anti-tumor efficiency of immunotherapy for PDAC.