Pharmacy

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Research from the School of Pharmacy. The website may be found at https://www.ndsu.edu/pharmacy/

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    Chemical Modification of Bovine Milk Exosomes, the Biological Nanoparticles of the Future, as a Contrast Agent and Drug Delivery Vehicle
    (North Dakota State University, 2021) Pullan, Jessica Elaine
    Chemically derived nanoparticles are widely used across many applications. While they showed great promise when first discovered, the main hurdles, such as clearance and targeting, have yet to be overcome. A recently discovered class of biological nanoparticles have the potential to circumvent these disadvantages. Exosomes are biological nanoparticles (30 – 150 nm) excreted from most mammalian cells. While exosomes are typically involved in cellular signaling and traditionally removed from the body to be examined for biomarkers, this work combines chemical modifications and a biological particle for diagnostics and treatment of solid tumor cancer. Exosome involvement in cancer treatment has grown over the past ten years with the encapsulation of RNA, proteins and traditional chemotherapeutics. However, this work takes these ideas and drives them into the future by using bovine milk derived exosomes as (1) an ultrasound contrasting agent and (2) a targeted and triggered chemotherapeutic drug delivery vehicle. As an ultrasound contrast agent, raw and pasteurized bovine milk exosomes were tested and found to be capable of echogenicity without altering the ability to identify key features of the exosome, including the presence of CD63 and miRNA. In the second part of this work a chemically synthesized, hypoxia responsive lipid and a tumor penetrating and targeting peptide, iRGD were integrated into the lipid bilayer of the exosome for chemotherapeutic drug delivery. These modified exosomes were characterized using a variety of techniques, including a novel adhesion assay, atomic force microscopy, and high-resolution transmission electron microscopy. The functional capacity of the modified exosomes to deliver doxorubicin to Triple Negative Breast Cancer (TNBC) cells was also evaluated using a combination of cellular internalization and cytotoxicity assays in both monolayer and 3D spheroid cultures. Overall exosomes have the ability to be chemically modified in a variety of ways, opening a door to a new approach to nanoparticle drug delivery and targeted imaging.
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    Determination of Growth Inhibitory Effect of Iminodibenzyl Against Breast Cancer
    (North Dakota State University, 2021) Shah, Harshit Pareshbhai
    Breast cancer arises from the culmination of complex process enclosing multiple gene modifications such as cyclooxygenase-2 (COX-2). It catalyzes arachidonic acid (AA, downstream ω-6 polyunsaturated fatty acid (ω-6 PUFA)) metabolism to cancer-promoting prostaglandin E2 (PGE2). Hence, COX-2 inhibition was considered an ideal strategy to inhibit the cancer progression. However, COX-2 inhibitors are no longer advised for cancer management due to life threatening cardiovascular adverse events. Recently, we found that inhibition of delta-5-desaturase (D5D, enzyme catalyzing di homo-gamma-linolenic acid (DGLA) metabolism to AA) in breast cancer cells by siRNA/shRNA caused the diversion of DGLA metabolism from PGE2 to anticancer metabolite 8-hydroxyoctanoic acid (8-HOA). But, the approach of using siRNA/shRNA was limited by endonucleases mediated physiological degradation and inability to cross the cell membrane. Therefore, to overcome the limitation and to stimulate DGLA metabolism towards anti-cancer activity, small molecule D5D activity inhibitor Iminodibenzyl was identified. Here, we have hypothesized that Iminodibenzyl could inhibit the DGLA metabolism by inhibiting the D5D activity, and simultaneously overexpressed COX-2 in breast cancer cells would peroxidize the accumulated DGLA to an anti-cancer metabolite 8-HOA. To achieve the research goal, we have performed various in vitro and in vivo studies (orthotopic breast cancer model). From these studies, we noted that Iminodibenzyl could alter DGLA metabolism to anti-cancer metabolite 8-HOA in 4T1 and MDA-MB-231 breast cancer cells. After treating cancer cells with the combination of Iminodibenzyl and DGLA, a significant increase in apoptosis was observed through the caspasedependent mechanism, which was validated by pretreating cells with nonspecific caspase inhibitor Z-VAD-FMK. Additionally, a significant reduction in HDAC activity and β-Catenin was observed, which might have reduced cancer cell survival fraction and proliferation. We believe that all the above mechanisms affected by the combination might have reduced the cancer growth resulting in significant reduction in tumor size. Additionally, combination treatment also reduced lamellipodia and filopodia, and EMT markers resulting in reduction in cancer cell migration as visible from larger wound size and less number of metastatic nodules. Hence, all the above findings provide evidence about the efficacy of Iminodibenzyl to shift the DGLA metabolism producing anti-cancer activity in breast cancer cells.
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    GLUT-1 Targeted Gene Delivery to Brain for the Treatment of Alzheimer's Disease
    (North Dakota State University, 2021) Arora, Sanjay
    Alzheimer’s disease (AD) is a neurodegenerative disorder resulting in debilitating dementia with progressive loss of motor functions. Genetic modulation of neurotrophic factors and apolipoprotein E (ApoE) have emerged as powerful strategies offering preventive and protective effect against AD pathophysiology. Brain derived neurotrophic factor (BDNF), apolipoprotein E2 (ApoE2) and vgf (non-acronymic) which play a major role in neuronal plasticity, synapse formation, amyloid-beta regulation and cognition, are found to be reduced in the brain of AD patients. However, delivery of such large polar proteins (BDNF, ApoE2 and vgf) across blood brain barrier (BBB) is one of the most challenging tasks. Therefore, in this study, we developed and optimized liposomal nanoparticles capable of delivering gene encoding for BDNF, ApoE2 and vgf to the brain in a targeted manner. These nanoparticles were surface modified with glucose transporter-1 targeting ligand (mannose) and various cell penetrating peptides to promote selective and enhanced delivery to brain. Dual-modified nanoparticles demonstrated homogenous size between 150-200 nm with positive zeta potential. These nanoparticles demonstrated ∼50% higher transport across in vitro BBB model and showed significantly higher transfection of encapsulated pDNA in bEnd.3 cells, primary astrocytes and neuronal cells. Surface functionalized nanoparticles also demonstrated significantly higher transport (∼7% of injected dose/gram of tissue) and gene transfection (1.5 - 2 times higher than baseline level) across BBB following single intravenous administration in C57BL/6 mice without any signs of toxicity. Furthermore, liposomal nanoparticles encapsulating pBDNF tested in early (6-months) and advanced stages (9-months) of transgenic APP/PS1 mouse model of AD showed good functional efficacy. The dual-modified nanoparticles enhanced BDNF expression by ~2 times and resulted in >40% (p<0.05) reduction in toxic amyloid-beta in 6- and 9- months old APP/PS1 mice brains compared to their age-matched untreated controls. Plaque load was reduced ~7 and ~3 times (p<0.05), respectively, whereas synaptic proteins, synaptophysin and PSD-95, were found to be increased by >90% (p<0.05) in both age groups of transgenic mice following BDNF treatment using dual-modified nanoparticles in comparison to their age-matched controls. Moreover, no untowardly adverse effects were observed throughout treatment, suggesting a safe and effective strategy for treatment of AD pathophysiology.
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    Understanding the Role of Receptor for Advanced Glycation Endproducts (RAGE) in Pancreatic Cancer and Melanoma
    (North Dakota State University, 2021) Taneja, Sakshi
    In this project we study the role of RAGE in the melanoma and pancreatic cancer progression. Based on published studies, we hypothesized that RAGE localization in melanoma varies with different cellular architectures. To test this hypothesis, we utilized an in vitro spheroid model and a lung colonization mice model to compare the RAGE localization in 3D architecture vs 2D monolayer culture. RAGE was found at the cell surface in WM115 and B16F10 spheroids, whereas RAGE is mostly distributed intracellularly in WM266. We also observed that RAGE is present at the surface of B16F10 melanoma cells within tumor nodules in the lungs of mice colonized with B16F10 cells. Previously, our group has demonstrated that RAGE promotes pancreatic tumor cell survival under normoxic conditions, upon gemcitabine administration. Hypoxia is also associated with increased tumor aggressiveness. Based on published reports, we hypothesized that RAGE upregulation under hypoxic conditions contributes to autophagy and migration in pancreatic cancer cells. We observed that autophagy decreases after RAGE inhibition by FPSZM1. Moreover, we observed decreased cell migration after RAGE blockage, indicating that RAGE also mediates migration under hypoxia. We also investigated Advanced Glycation Endproducts (AGEs) on proliferation and migration of pancreatic cancer cells. Based on published reports, we hypothesized that RAGE activation by AGEs contributes to the proliferation and migration in pancreatic cancer cells. We employed ribose modified BSA to activate RAGE in the murine KPC 5517 pancreatic cancer cell line. We observed that AGE-treated samples showed significant increase in migration but no change in proliferation. As RAGE is involved in the progression of melanoma and pancreatic cancer, our results will help researchers to better understand the biology of RAGE. Our research can help to design RAGE-specific antibodies and inhibitors that could target RAGE more effectively. Moreover, our findings on AGE-RAGE interactions, and on the role of RAGE in pancreatic cancer progression under hypoxia, may contribute to reduce the progression of pancreatic cancer. Our results showing that a RAGE inhibitor can reduce autophagy and migration of pancreatic tumor cells, suggest that FPS-ZM1 could be utilized as a potential therapeutic aid for the treatment of pancreatic cancer.