Pharmacy Doctoral Work

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Estrogen Receptors Signaling in Airway Smooth Muscles: Role in Intracellular Calcium Regulation and Contractility
(North Dakota State University, 2021) Sangeeta, Bhallamudi
Epidemiological evidence suggests higher incidence and severity of asthma in pre-menopausal women and aging men, suggesting a role of sex steroids, especially estrogen. In this regard, it is not clear whether specific estrogen receptors (ERα and ERβ) play differential roles or whether there is any imbalance in their normal signaling during asthma. Airway smooth muscle (ASM) cell is of contractile phenotype which is involved in contractions and airway hyperresponsiveness. Therefore, this research focused to understand ER signaling in the context of airway hyperresponsiveness and bridge the gaps in knowledge about the role of ERs in human ASM cells. The first aim demonstrate the long-term differential signaling of ERs in the regulation of [Ca2+]i handling in the human ASM. It was found that ERα activation increases the [Ca2+]i response in baseline conditions, while ERβ activation has neutral effect. Moreover, the differential signaling of ERs is more evident in asthma or inflammation where ERβ activation decreases the [Ca2+]i response in the presence of inflammation while ERα increases it. Further elucidation of the mechanisms of their signaling on [Ca2+]i suggests that ERβ results in decreased [Ca2+]i response through increased SERCA2 expression and function, inhibition of pathways involved in activating the voltage gated LTCC and maintenance of the morphology of mitochondria. In the next aim, it was found that ERβ causes a potentiation of the activity of β2-AR which leads to an increase in cAMP. Also, ERβ is found to be involved in dephosphorylation of contractile apparatus ultimately leading to bronchodilation. Presenting a contrasting picture, ERα causes an increase in pro-contractile machinery such as RhoA activity and phospho-MYPT leading to increased overall contractility in ASM cells. To confirm these in vitro findings in the presence of other structural and immune cells involved in inflammation, I have further evaluated the ex vivo and in vivo roles of ER signaling in airways. Interestingly, ERβ was found to be protective for the airways while ERα further aggravated the contractility of the airways. These novel findings of ER signaling in the context of contractile mechanisms of the airways can be utilized in designing novel therapeutics for bronchodilation.
BKCa-IP3R Decoupling in Hypertension
(North Dakota State University, 2022) Niloy, Sayeman Islam
Hypertension is a significant risk factor for cardiovascular diseases and a leading cause of worldwide morbidity and mortality. Dysregulation of intracellular Ca2+ in vascular smooth muscle (VSM) cells is one major contributor to the development of vascular hypercontractility and remodeling in hypertension. Plasma membrane (PM)-localized large-conductance, Ca2+-activated K+ (BKCa) channels prevent hypercontractility through membrane hyperpolarization in response to vasoconstrictor-induced activation of inositol trisphosphate receptors (IP3Rs), localized on the sarcoplasmic reticulum (SR). However, loss of close contact or coupling between BKCa and IP3R may diminish the BKCa-mediated protection against hypercontractility and hypertrophy and contribute to the development of hypertension. The overall goal of this study was to understand the role of BKCa-IP3R coupling in the development of vascular hypercontractility and remodeling. I used a hypertensive animal model, spontaneously hypertensive rat (SHR), to study the impact of the loss of this coupling. My hypothesis was that there is a loss of communication between the IP3 receptors and the BKCa channels in SHR VSM cells leading to reduced BKCa current after IP3R activation. My first objective was to determine the role of functional coupling of BKCa and IP3R in vascular hypercontractility and hypertrophy development. Based on the findings, one can conclude that in SHR mesenteric VSM cells, there is a loss of functional IP3R-BKCa coupling, and it might be involved in vascular hypercontractility and hypertrophy. My second objective was to examine and compare the molecular coupling of BKCa and IP3R between normotensive and hypertensive rats. My data suggest that the molecular connection between BKCa and IP3R is disrupted in SHR VSM cells. My results also suggest that this loss of connection is not due to downregulation of junctophilin-2 (JPH2) but may be due to defective tethering of JPH2 to the PM. Together, this research provides an improved understanding of the crucial roles played by BKCa-IP3R coupling in hypertension. An understanding of ion channel coupling under disease conditions may provide relevant caveats where BKCa channels are considered a therapeutic target. I expect that the knowledge gained from my studies will fundamentally advance the field of ion channel-based therapeutics, especially in cardiovascular disorders.
Understanding the Role of Rage in Cell Adhesion
(North Dakota State University, 2022) Thiyagarajan, Swetha
The Receptor for Advanced Glycation Endproducts (RAGE) is a mammalian specific cell surface receptor. RAGE consists of three extracellular domains (V, C1, and C2), a transmembrane domain, and an intracellular cytoplasmic tail. RAGE has a significant role in human pathogenesis, including neurodegenerative diseases, diabetic complications, and certain cancers. Deregulation of cell adhesion is one of the contributing cellular events common in many of the above listed human pathologies and might be mediated via RAGE signaling. In our study, we aimed to understand the role of RAGE in cell adhesion and to define the importance of the different domains of RAGE in mediating this phenomenon. For this study, a protein engineering approach was used to express full-length RAGE (FL-RAGE) and a panel of domain deletion constructs ((ΔV-, ΔC1-, ΔC2-, DN-, TmCyto-) RAGE) of the receptor. The necessary expression constructs were assembled in the pcDNA3 vector, and the RAGE variants were expressed in HEK293 cells. The expression and cellular localization of RAGE in HEK 293 cells were analyzed using Western blot, immunofluorescence microscopy, and flow cytometry techniques. Our results show that the cytoplasmic domain of RAGE was sufficient to contribute to cell adhesion to the extracellular matrix to a level comparable to that of the FL-RAGE expressing cells. The current mechanistic model suggests that RAGE signaling is initiated by ligand binding to the extracellular region, followed by conformational changes in the intracellular domain. Subsequently, this conformation change leads to the recruitment of RAGE-interacting proteins on the intracellular side of the plasma membrane. However, in this thesis, we present evidence of an alternative mechanism of RAGE signaling possibly involving the translocation of RAGE into the nucleus. The results from our study suggest an alternative model for RAGE signaling and will help to better understand RAGE signaling in pathophysiological conditions. Our results could contribute to the development of new small molecule drugs targeting intracellular RAGE or the intracellular RAGE domain as a novel approach for inhibiting RAGE signaling.
Kisspeptins: Airway Remodeling in Asthma
(North Dakota State University, 2022) Borkar, Niyati
Asthma is a chronic respiratory disorder that affects people of all ages. Sex and gender disparity in asthma is recognized and suggests a modulatory role for sex steroids, particularly estrogen. However, there is a dichotomous role of estrogen in airway remodeling, making it unclear whether sex hormones are protective or detrimental in asthma and suggesting a need to explore mechanisms upstream or independent of estrogen. Kisspeptins (Kp) are a novel peptide existing upstream of sex steroids and function as a crucial regulator of puberty. Kp via KISS1R are further implicated in the sex steroid biogenesis via action on the gonadotrophins. Therefore, this research hypothesizes that kisspeptin (Kp)/KISS1R signaling serves this role, thereby regulating airway remodeling and airway hyperresponsiveness (AHR).Airway smooth muscle (ASM) is a key structural cell type that contributes to remodeling in asthma. In the first aim, I report novel data indicating that Kp and KISS1R are expressed in human airways, especially ASM, with lower expression in ASM from women compared with men and lower in patients with asthma compared with people without asthma. My second aim discusses the functional mechanisms of Kp/KISS1R signaling on majorly proliferation, and in part ECM deposition studies. Proliferation studies show that Kp-10, mitigates PDGF-induced ASM proliferation. Pharmacological inhibition and shRNA knockdown of KISS1R increased basal ASM proliferation, which was further amplified by PDGF. The anti-proliferative effect of Kp-10 in ASM was mediated by inhibition of MAPK/ERK/Akt pathways, with altered expression of PCNA, C/EBP-α, Ki-67, cyclin D1, and cyclin E leading to cell cycle arrest at G0/G1 phase. ECM studies show that administration of Kp-10 can mitigate PDGF- and TGFβ- induced increase in ECM remodeling gene and protein expression, such as collagens and fibronectins. To corroborate my in vitro findings, I have further performed in vivo studies utilizing Kp-10 (a cleaved peptide of parent kisspeptin) in mixed allergen-induced mouse models of asthma. I found that Kp-10 was able to mitigate asthma by significantly improving airway structural, morphological and lung function parameters. Overall, I demonstrate the importance of Kp/KISS1R signaling in the ASM as a potential therapeutic avenue to blunt remodeling in asthma.
Apelin-APJ Signaling in Hypertensive Coronary Arteries
(North Dakota State University, 2022) Anto, Santo Kalathingal
Apelin is an endogenous ligand for APJ receptors, which are highly expressed throughout the cardiovascular system, including coronary arteries. Apelin causes endothelium‐derived nitric oxide (NO) –dependent relaxation of coronary arteries under physiological conditions, but little is known about regulation of coronary vasomotor tone by apelin under pathological conditions. This research addresses the critical gap in understanding of the apelin signaling in coronary circulation under normal and pathological conditions. Evidence from this study suggests that apelin could not provide beneficial vasodilatory effects in hypertensive coronary arteries. Moreover, apelin impairs endothelium-dependent relaxations to vasodilator, acetylcholine. Impaired apelin-APJ signaling in hypertensive coronary arteries is possibly through defective production or release of NO from coronary endothelial cells rather than inhibiting effects of NO in coronary arterial smooth muscle cells. My next aim was to understand the mechanisms involved in the loss of apelin response in hypertensive arteries. The results suggested that the APJ receptor signaling via GRK2 pathway is possibly responsible for the impaired apelin response in hypertensive coronary arteries. Interestingly, APJ receptor biased agonist, CMF-019, -induced relaxation in hypertensive coronary arteries and showed no effects on vasodilatory response to acetylcholine. My results also suggest the possible impairment of PI3K/AKT/eNOS pathway mediated by GRK2 activation in hypertensive coronary arteries. My final aim was to check whether apelin signaling is impaired in coronary arteries only under hypertensive conditions or if it occurs in other disease conditions. My data suggests that apelin signaling is impaired in coronary arteries exposed to cigarette smoke extract (CSE), a model for secondhand smoke exposure. Interestingly, similar to hypertensive coronary arteries, apelin lost its beneficial vasodilatory effects possibly through the GRK2 activation in CSE treated coronary arteries. Overall, this research provides evidence that apelin behaves differently under physiological and pathological conditions. As a point of fact, apelin not only lost its beneficial effects but also might have negative effects under pathological conditions such as hypertension and secondhand smoke exposure. I anticipate that the results from this approach will be useful in improving the therapeutic strategies with apelin and other APJ receptor agonists that are aimed to alleviate different cardiovascular disorders.
Development of Nano-Architecture Systems for High-Efficiency Tumor-Targeted Drug Delivery
(North Dakota State University, 2021) Mamnoon, Babak
Breast cancer is the most common malignancy and the second leading cause of death among women in the United States. The commonly used breast cancer treatment strategies include surgery, chemotherapy, radiation, and hormonal therapy. Since chemotherapeutic agents do not adequately differentiate between normal and cancerous cells, systemic toxicity and adverse effects associated with these anticancer drugs limit their therapeutic efficacy. In addition, uncontrolled cell proliferation and insufficient blood supply produce low oxygen partial pressure or hypoxia in almost all solid tumors. Hypoxia increases cancer cell survival through aggressiveness, metastasis, and resistance to chemotherapy, leading to poor clinical outcomes. Targeted drug delivery nanoparticles can significantly reduce off-target toxicity of chemotherapy by selectively targeting tumor tissues. Polymersomes are self-assembled drug-encapsulated polymeric nanoparticles in which an aqueous core is enclosed by a bilayer membrane. To attain the appropriate therapeutic efficacy, polymersomes need to rapidly release their anticancer drug at the tumor sites. To fulfill this requirement, stimuli-responsive polymersomes have been developed. Since most of the tumors have hypoxic areas inside, hypoxia-responsive polymersomes are one of the most effective drug delivery vehicles for cancer treatment. To prepare targeted drug delivery systems, functionalizing polymersomes with specific ligands intended to be recognized by the receptors of the cancer cells, is the most common strategy. Herein, we designed three distinct hypoxia-responsive polymersomes for targeting breast cancer tissues. More than 80% of breast cancers express estrogen receptor (ER-positive), and about 15-25% of them do not express any receptors (triple-negative). Hence, we decorated our polymersomes with three different ligands including estradiol and endoxifen for targeting ER-positive breast microtumors, and iRGD peptide for targeting triple-negative breast tumors.
Targeted Three-Way-Junction RNA Nanoparticle Reprogrammed Cyclooxygenase-2 Catalyzed Dihomo-ϒ-Linolenic Acid Peroxidation Pattern in Lung Cancer
(North Dakota State University, 2021) Pang, Lizhi
As the second most common cancer and the first leading cause of cancer deaths, lung cancer attracts much research attention. Since cyclooxygenase-2 (COX-2) is overexpressing in nearly 80% of lung cancer patients, COX-2 inhibitors may benefit cancer patients via breaking the COX-2/arachidonic acid (AA)/ prostaglandin E2 (PGE2) axis, which is highly relevant to cancer progression. However, the COX-2 inhibitors fail to improve survival of cancer patients in clinical studies. To effectively take advantage of COX-2 overexpression in lung cancer, in this study, we advanced a novel strategy, instead of direct COX-2 inhibition, redirecting COX-2 catalyzed dihomo-γ-linolenic acid peroxidation by knocking down delta-5-desaturase (D5D) in lung cancer cells. We found that the D5D siRNA knockdown could repress the AA and PGE2 formation in lung cancer cells. While a distant free radical byproduct, 8-hydroxyoctanoic acid (8-HOA) was derived from DGLA by COX-2. Both exogenous 8-HOA and DGLA-derived endogenous 8-HOA resulted in inhibition of proliferation, survival, migration but activation of apoptosis in lung cancer cells. We believe that the inhibitory effect of 8-HOA lung cancer is possibly due to histone deacetylase (HDAC) and YAP1/TAZ inhibition. Additionally, we demonstrated the synergistic effect between 8-HOA and first-line chemo cisplatin on lung cancer. To improve the efficiency of D5D inhibition, we used innovative RNA nanotechnology to specifically deliver D5D siRNA to lung cancer in vitro and in vivo. The D5D siRNA was harbored by 3 way-junction (3WJ) RNA nanoparticle along with epithelial cell adhesion molecule (EpCAM) aptamer as targeting module, and Alexa 647 as imaging module. By using lung cancer and normal lung epithelial cell models, we demonstrated that 3WJ-EpCAM-D5D siRNA nanoparticles could suppress lung cancer cell growth by promoting 8-HOA formation in a COX-2 dependent manner. The EpCAM aptamer ensured the specific in vivo delivery of RNA nanoparticles to lung tumors, avoiding damage to other tissues and toxic/off-target effect. 3WJ-EpCAM-D5D siRNA nanoparticle significantly inhibited xenograft tumor growth in nude mice by regulating apoptosis, metastasis, and proliferation. Overall, our strategy generated an effective and safer outcome and paved the road to COX-2-based precise medicine for lung cancer therapy.
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.
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.
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.
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.
Antibiotic Releasing Bone-Void Filler for the Treatment of Osteomyelitis: An Approach to Treat Infection and Aid Bone Regeneration
(North Dakota State University, 2020) Hasan, Mohammad Raquibul
Osteomyelitis or bone infections remain very difficult to treat despite advances in treatment regimens and surgical technics. The bone microenvironment and compromised vasculature in addition to infected prosthesis and implants that were put in the bone during prior surgery impedes the antibiotic partition into the bone from systemic therapy in many cases. Treatment often includes surgical debridement of the infected bone and surrounding tissue, removal of implants, systemic antibiotic therapy accompanied with antibiotic containing bone void filler, in most cases polymethylmethacylate (PMMA) based bone cement. Unfortunately, PMMA has many associated problems, including non-biodegradability, inconsistent antibiotic release, and a surface susceptible to bacterial biofilm growth, ultimately necessitating removal and causing recurrent infections. Thus, recent studies have focused on designing novel bone void filling materials to deliver antibiotics and to support bone regeneration. There are two parts to designing a successful bone void filling device/material:(1) local release antibiotic for infection treatment and (2) development of a bone graft substitute to support bone regrowth. In this study, antibiotic releasing bone void filler (ABVF) putty formulations have been designed and tested. Different formulations were examined in this dissertation to describe the three components of the putty formulation - polymer, drug, and substrate. In the first formulation, different custom-made polymers were used to control drug release; Pro Osteon, a hydroxyapatite (HA) and calcium carbonate based bone graft substitute was used to provide support for bone growth. Finally, vancomycin was used as the antibiotic as it is clinically used to treat Staphylococcus aureus, the primary cause of osteomyelitis. In second formulation, commercially available and clinically used polymers, poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL) and, polyethylene glycol (PEG), were used to make the ABVF putty along with Pro Osteon and vancomycin. In the subsequent formulations, delivering combination antibiotics - vancomycin and rifampicin - to treat biofilm infections and, using bioglass (BG) as the substrate for faster bone regrowth were explored; PLGA, PCL and PEG constituted the polymer matrix. The ABVF putty formulations were customizable in terms of three primary components: polymers, bone graft substitutes, antibiotics. Ultimately, these were successful in curing infection and providing bone growth support.
Modifications of Recombinant Spider Silk Protein for Various Biomedical Applications
(North Dakota State University, 2020) Mulinti, Pranothi
Silk is a natural protein produced by members of the class Arachnida (over 30,000 species of spiders) and by several worms. Silk-based materials have been investigated for medical and biotechnological applications for many years. Although silkworm silk has been studied extensively because of ready availability of the protein, lately the advancements in recombinant technology has made production of spider silk proteins increasingly available. Due to the characteristics like biocompatibility, biodegradability and mechanical strength, silk is highly desirable as a biomaterial for medical purpose. Along with this, techniques for functionalization, has further aided in the development of silk into highly sophisticated material for advanced applications. The main objective of this thesis has been to investigate novel strategies for functionalization of the recombinant spider silk protein Masp2. Two distinct approaches were used, chemical modification and genetic fusion. In the first modification, we created an infection responsive silk nanospheres by chemically grafting a thrombin sensitive peptide to the silk protein encapsulating antibiotic. These particles were then evaluated for in vitro infection responsive drug release and antimicrobial activity. From these assessments, we found that these particles can release the drug effectively in the presence of infection providing the evidence that these particles are enzyme responsive and can be used to formulate targeted drug release. In the second modification, spider silk was genetically modified with a heparin binding peptide to create a fusion protein which can prevent both thrombosis and infection simultaneously. This fusion protein was evaluated for its heparin binding ability and anticoagulant properties in its solution form. Furthermore, due to the similarity in structure of HBP with antimicrobial peptides, it is predicted that the fusion protein will also show antimicrobial property. After establishing these properties, next this fusion protein was utilized as a coating for hemodialysis catheter. Deposition of coating was evaluated after which anticoagulant and anti-infective properties of the protein as a coating material was investigated. This thesis provides evidence of successful production of a recombinant silk-based biopolymer that can be chemically and genetically embedded with a various functional motif to create a hybrid product for different applications.
Drug Delivery Systems for Treatment of Diabetes Mellitus
(North Dakota State University, 2019) Sharma, Divya
Daily injections for basal insulin therapy are far from ideal resulting in hypo/hyperglycemic episodes associated with fatal complications in type-1 diabetes patients. The purpose of this study was to develop a thermosensitive copolymer-based in situ depot forming delivery system to provide controlled release of insulin for extended duration following a single subcutaneous injection, closely mimicking physiological basal insulin requirement. Size and nature of the incorporated therapeutic were observed to affect the release profile of insulin. Modification with zinc and chitosan preserved thermal, conformational, and chemical stability of insulin during the entire duration of storage (up to 9 months at 4 °C) and release (up to 3 months at 37 °C). In vivo, daily administration of long-acting insulin, glargine, resulted in fluctuating blood glucose levels between 91 – 443 mg/dL in type 1 diabetic rats. However, single administration of oleic acid-grafted-chitosan-zinc-insulin complexes incorporated in copolymer formulation demonstrated slow diffusion of insulin complexes maintaining peak-free basal insulin level of 21 mU/L for 91 days. Sustained release of basal insulin also correlated with efficient glycemic control (blood glucose <120 mg/dL), prevention of diabetic ketoacidosis and absence of cataract development, unlike other treatment groups. The suggested controlled basal insulin delivery system has the potential to significantly improve patient compliance by improving glycemic control and eliminating life-threatening diabetes complications. Furthermore, oleic acid-grafted-chitosan (CO) nanomicelles were investigated as a non-viral vector to deliver plasmid DNA encoding short hairpin RNA (shRNA) against pro-inflammatory cytokines to adipose tissue macrophages and adipocytes for the treatment of insulin resistance. Nanomicelles modified using mannose (COM) and adipose homing peptide (AHP) (COA) showed significantly higher uptake and transfection efficiency in inflamed macrophages- adipocytes co culture owing to glucose transporter-1 and prohibitin receptor mediated internalization, respectively. Ligand modified nanomicelles loaded with shRNA against tumor necrosis factor alpha (COM-TNFα) and monocyte chemoattractant protein-1 (COA-MCP1) demonstrated significant attenuation of pro-inflammatory cytokines and improved insulin sensitivity and glucose tolerance in obese-diabetic mice for six weeks post treatment with single dose of optimized formulation. Overall, chitosan nanomicelles mediated targeted gene therapy can help attenuate inflammation, the chief underlying cause of insulin resistance, thereby helping reverse the progression of diabetes.
A Nano-Sized Approach to Exploiting the Pancreatic Tumor Microenvironment
(North Dakota State University, 2020) Confeld, Matthew Ian
Making up just over 3% of all new cancer cases in the United States, pancreatic cancer is not inherently a common malignant disease. Yet, it continuously is shown to be one of the most lethal and common causes of cancer death. Early detection is critical among all cancer types. However, oncologists and researchers have struggled to find effective strategies or tests to detect cancer of the pancreas early on in development. Thus, the cancer is often found late stage and requires significant chemotherapy intervention. These multi-drug treatment cocktails have shown benefit, but only in added months and not years to a patient’s life. Significant adverse effects often limit the full effective doses of treatment. In order to limit these adverse effects, as well as increase the effectiveness of treatment, we have designed, optimized, and tested unique drug carriers known as polymersomes. Using characteristics of the localized environment surrounding pancreatic tumors and the cells found therein, we created targeted therapies that are responsive and relatively selective toward cancerous cells. Herein, are found two distinct polymersomes. The first, is a low oxygen reactive drug carrier with an additional small peptide molecule that is able to penetrate dense tumor tissue and has shown decreased tumor growth of as much as 260% as compared to control samples in an animal model of pancreatic cancer. The chemical make-up of this polymersome allows for extended circulation time and a high accumulation at the tumor site. A second design, uses an intracellular enzyme to destabilize the polymersomes’ structure, which in turn, releases a selected chemotherapy drug near its intended site of action. This strategy, has shown a 10 fold increase in potency of the chemotherapy drug, as compared to when the drug is given alone and showed decreased toxicity to non-cancerous cells. It is certain that thoughtful drug delivery strategies and not just drug molecule design will be instrumental in the paradigm shift of pancreatic cancer from likely death to survival.
The Role of the Gut Microbiota in Sustained Weight Loss Following Roux-en-Y Gastric Bypass Surgery
(North Dakota State University, 2018) Fouladi, Farnaz
Roux-en-Y Gastric Bypass (RYGB) surgery is one of the most effective approaches for the treatment of severe obesity. Despite substantial weight loss following RYGB, a considerable proportion of patients experience weight regain or insufficient weight loss. The proposed research aimed to investigate the role of the gut microbiota in weight regain or suboptimal weight loss following RYGB. The gut microbiota composition in post-RYGB patients who experienced successful weight loss (SWL, n=6), post-RYGB patients who experienced poor weight loss (PWL, n=6), and non-surgical controls (NSC, n=6) who were age- and BMI-matched to the SWL group (NSC, n=6) were characterized through 16S rRNA gene sequencing. To further investigate the impact of the gut microbiota on weight profile, human fecal samples were transplanted into antibiotic-treated mice through oral gavage. Food intake and body weight were measured at weekly intervals for a month. At five weeks following colonization mice were randomly switched to a Western Diet or maintained on a normal diet. The results showed that Lactobacillales, Enterobacteriales, and Verrucomicrobials were enriched in both surgical groups compared to the NSC group. No significant difference was observed in the gut microbiota composition between PWL and SWL patients. However, transfer of the gut microbiota from human patients into antibiotic-treated mice resulted in significantly greater weight gain in PWL recipient mice compared to SWL recipient mice at four weeks following colonization (15.03±2.59% versus 7.88±1.28%, F(2,41)=4.01 p=0.026). We found that Barnesiella, Gordonibacter, Parasutterella, Clostridium cluster XVIa were effectively transferred from humans to mice and were associated with weight gain in recipient mice. Interestingly, Barnesiella that tended to be higher in PWL humans was also significantly higher in PWL recipient mice compared to SWL and NSC recipient mice. All three groups of recipient mice gained weight when they were placed on the Western Diet regardless of human donor group. In summary, the results indicate that the gut microbiota are at least functionally different between PWL and SWL patients. Some taxa may contribute to weight gain after surgery. Future studies will need to determine the molecular mechanisms behind the effects of the gut bacteria on weight regain after RYGB.
Understanding the Role of the Receptor for Advanced Glycation End-Products (Rage) in Pancreatic Cancer
(North Dakota State University, 2019) Swami, Priyanka
Expression of the Receptor for Advanced Glycation End Products (RAGE) and is upregulated in a several cancers. Based on published studies, we hypothesized that RAGE, when overexpressed in pancreatic cancer cells, will promote cell proliferation and migration. To study the role of RAGE in pancreatic cancer, we selected the human pancreatic cancer cell-line PANC-1, and stably transfected the cells with full length RAGE to generate model cell-lines that overexpress RAGE. We obtained two cell-lines PANC-1 FLR2 and PANC-1 FLR3 and examined the influence of RAGE on cellular properties. A significant increase in proliferation but a reduction in migratory abilities of PANC-1 FLR2 and PANC-1 FLR3 cells was observed. The increase in proliferation and reduction in migration was reverted upon knockdown of RAGE in PANC-1 FLR2 cells with siRNA specific for RAGE. The reduction in migration was supported by the reduced levels of vimentin and several integrins in RAGE transfected cells. Furthermore, we observed a downregulation in FAK, AKT, ERK1/2 and NF-κB activity. Growing evidence supports that RAGE is essential for pancreatic cancer progression. It has also been shown that RAGE facilitates pancreatic tumor cell survival by enhancing autophagy and inhibiting apoptosis. The goal of our study was to determine the effect of RAGE inhibition during gemcitabine chemotherapy on the growth of pancreatic tumor. Hence, we investigated the effect of RAGE inhibitors and their combination with gemcitabine in an orthotopic mouse model of pancreatic cancer using mouse pancreatic cancer cell-line KPC 5508. We used two RAGE inhibitors, an anti-RAGE monoclonal antibody (IgG2A11) and a small molecule RAGE inhibitor (FPS-ZM1). We observed a significant reduction in tumor weights of the mice treated with the combination of IgG2A11 and gemcitabine as compared to gemcitabine alone treated mice. The reduction in tumor growth was accompanied with increase in p62 levels (marker of autophagy) and increase in levels of cleaved PARP (marker of apoptosis). We also observed reduction in HMGB1 and phosphorylation levels of ERK1/2 in tumors from the group treated with the combination as compared to the gemcitabine alone treated group.
Evaluation of Smart Polymers for Controlled Release Delivery Systems
(North Dakota State University, 2019) Lipp, Lindsey
Our goal was to develop a smart polymer, controlled release delivery system and evaluate its capabilities for use with salmon calcitonin and rivastigmine. Thermosensitive and phase sensitive smart polymers were evaluated for their potential as controlled release delivery systems. Thermosensitive triblock copolymers were synthesized with increasing lactide to glycolide ratios of 3.5:1, 4.5:1, and 5:1. Characterization was via analytical techniques including proton nuclear magnetic resonance, gel permeation chromatography, critical micellar concentration, sol-gel transition test tube inversion, and cellular biocompatibility assay. Only the 5:1 lactide to glycolide copolymer transitioned into gel at body temperature. Release duration in vitro was 70 days when salmon calcitonin was incorporated at 40% (w/v) in 5:1 thermosensitive copolymer while retaining the native conformation of salmon calcitonin as analyzed via micro bicinchoninic acid assay, circular dichroism and differential scanning calorimetry. Optimization of thermosensitive and phase sensitive copolymers for delivery of rivastigmine was extensively studied thereafter by comparing key variables of: rivastigmine hydrophobicity, polymer concentration, rivastigmine concentration, and depot volume. The optimal thermosensitive formulation was composed of 35% (w/v) copolymer at an injection volume of 0.5 ml containing 40 mg/ml of rivastigmine base. The release of rivastigmine base was observed for ~16 days in a zero-order fashion. For phase sensitive polymer, we found the best formulation after optimization was that of 5% (w/v) 50:50 poly(lactic-co-glycolic acid) in 95:5 benzyl benzoate to benzyl alcohol with rivastigmine base incorporated at 216 mg/ml. Release was observed over the course of ~42 days. In vivo testing was performed using the optimized phase sensitive smart polymer composed of 50:50 PLGA at 5% (w/v) in 95:5 benzyl benzoate with rivastigmine tartrate incorporated as a suspension. Using this formulation, we achieved controlled release for 7 days. Acetylcholinesterase activity was evaluated in the brains of the rats at different time points for all conditions. Acetylcholinesterase was inhibited during controlled release of rivastigmine by 42% in 7 days, compared to healthy controls. The results demonstrate that controlled release of rivastigmine was accomplished and shows promise as a method to increase dosing interval and improve quality of life for those suffering from Alzheimer’s Disease.
Dual-Functionalized Liposomes for Gene Delivery to Brain to Prevent and Treat Alzheimer’s Disease
(North Dakota State University, 2019) dos Santos Rodrigues, Bruna
Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder which lacks effective disease-modifying therapies. We have investigated the therapeutic potential of pDNA encoding apolipoprotein E2 (ApoE2), or nerve growth factor (NGF) by transporting pDNA across the blood brain barrier (BBB) and expressing the ApoE2 or NGF into brain, using brain-targeted liposomal nanoparticles for treatment of AD. We explored the neuroprotective functions of ApoE2 and survival-promoting properties of NGF through gene therapy as potential disease-modifying therapies for AD. We designed brain-targeted gene delivery systems with prolonged systemic circulation and enhanced cellular penetration by conjugating transferrin (Tf) ligand and cell-penetrating peptide (CPP) to liposome via DSPE-PEG phospholipid. In vitro characterization studies showed that the nanoparticles had homogeneous particle size, positive zeta potential and protected plasmid DNA against enzymatic degradation. Additionally, they exhibited low hemolytic potential and low cytotoxicity. Cellular uptake occurred in a time-dependent manner through multiple endocytosis pathways. Reporter gene transfection and consequent protein expression in different cell lines were significantly higher using CPP-Tf-liposomes compared to single modified liposomes. The ability of these liposomes to escape from endosomes can be an important factor which may have likely contributed to the high transfection efficiency observed. In vivo brain targeting efficiency of designed liposomes was evaluated using in vitro triple co-culture BBB model. Dual-modified liposomes efficiently crossed in vitro BBB and, subsequently, transfected primary neuronal cells. Increasing NGF expression in primary neuronal cells following treatment with liposomes increased the levels of pre-synaptic marker synaptophysin in vitro. PenTf-liposomes containing pDNA efficiently induced protein expression in the brain of mice. A dose response study was performed in order to select the appropriate dose of pNGF to induce significant NGF expression and, consequently, a therapeutic effect. Administration of PenTf-liposomes containing pNGF to APP/PS1 mice (aged 3 months) for four weeks (one injection per week) decreased the levels of toxic soluble and insoluble Aβ peptides. Additionally, the treatment stimulated cell proliferation and increased the levels of synaptic markers, synaptophysin and PSD-95. These data suggest the therapeutic potential of PenTf-liposome-mediated NGF gene therapy which can be considered as a candidate for treatment of AD.
Dual Functionalized Liposomes for Co-delivery of Anti-cancer Chemotherapeutics for Treatment of Brain Tumor
(North Dakota State University, 2019) Lakkadwala, Sushant
Glioblastoma is a hostile brain tumor associated with high infiltration leading to poor prognosis. Currently available treatment options are insufficient to increase median survival time. The combination therapy has emerged as an efficient way to deliver chemotherapeutics for treatment of glioblastoma. It provides collaborative approach of targeting cancer cells by acting via multiple mechanisms, thereby reducing drug resistance. However, the presence of selective and impermeable blood brain barrier (BBB) restricts the delivery of chemotherapeutic drugs into the brain. To overcome this limitation, we designed a dual functionalized liposomes by modifying their surface with transferrin (Tf) and a cell penetrating peptide (CPP) for receptor and adsorptive mediated transcytosis, respectively. In this study, we used various CPPs based on their physicochemical properties (TAT, penetratin, QLPVM and PFVYLI) and investigated the influence of insertion of CPP to Tf-liposomes on cytotoxic potential, cellular uptake, hemocompatibility and transport across the BBB both in vitro and in vivo. In addition, anti-tumor efficacy of dual functionalized liposomes was evaluated in vitro as well as in vivo. The liposomes were encapsulated with chemotherapeutics agents, doxorubicin and erlotinib for delivery to brain. Co-delivery of doxorubicin and erlotinib loaded Tf-CPP liposomes revealed significantly (p < 0.05) higher translocation more than 12 % across the co-culture endothelial barrier resulting in regression of tumor in the in vitro brain tumor model. The biodistribution of Tf-CPP liposomes demonstrated more than 10 and 2.7 fold increase in doxorubicin and erlotinib accumulation in mice brain, respectively compared to free drugs. Histological evaluation of tissue sections showed no signs of toxicity. In addition, Tf-Pen liposomes showed excellent antitumor efficacy by regressing ~90% of tumor in mice brain with significant increase in the median survival time (36 days). In conclusion, we have developed a high efficiency liposomal drug delivery carrier that can cross the BBB and co-deliver doxorubicin and erlotinib to desired target tumor site in vivo in mice, thereby 1) increased concentration of chemotherapeutics in brain, 2) regression in glioblastoma tumor size, 3) reducing the possibility of drug resistance in cancer cells, without eliciting undesired toxicity.

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