Pharmacy Doctoral Work

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The Potential Inhibitory Effect of Dihomo-Gamma-Linolenic Acid on Colon Cancer Cell Growth via Free Radical Metabolites in Cyclooxygenase-Catalyzed Peroxidation
(North Dakota State University, 2012) Gu, Yan
Cyclooxygenase (COX) can metabolize dihomo-γ-linolenic acid (DGLA) and arachidonic acid (AA) through free radical-mediated lipid peroxidation to form the anti-carcinogenic 1-series of prostaglandins and pro-carcinogenic 2-series of prostaglandins, respectively. Our previous studies had demonstrated that in ovine COX-mediated DGLA and AA peroxidation, there are common and exclusive free radicals formed through different free radical reactions. However, it was still unclear whether the differences are associated with the contrasting bioactivity of DGLA vs. AA. In order to investigate the possible association between cancer cell growth and the exclusive free radicals generated from COX/DGLA vs. COX/AA, we refined our combined spin-trapping/LC/MS method with solid phase extraction to characterize free radicals in their reduced forms in the human colon cancer cell line HCA-7 colony 29, which has a high COX-2 expression. For the first time, we were able to profile free radical formation in the experimental settings in which cell proliferation (via MTS assay) and cell cycle distribution (via PI staining) could be assessed. Our results showed that DGLA- and AA-derived exclusive free radicals, rather than prostaglandins, were closely associated with the opposing bioactivities of DGLA vs. AA. Due to rapid conversion from DGLA to AA via Δ-5 desaturase (D5D), the anti-proliferative effect of DGLA on cancer cell growth was limited. Thus, double doses of DGLA and D5D inhibitor were introduced. Among DGLA, double-dose DGLA, and combined DGLA/D5D inhibitor treatments, the latter exerted the most anti-proliferative effect on cancer cell growth and caused significant cell G2/M arrest. D5D knockdown cells (via siRNA transfection) were used to further investigate the possible mechanism underlying the anti-proliferative effect of DGLA on cancer cell growth. In addition, the combined DGLA/D5D Iv inhibitor treatment increased the susceptibility of cancer cells to the chemotherapy drug 5-fluorouracil. In D5D knockdown cells, DGLA and 5-fluorouracil exerted greater effects on cell growth inhibition and cytotoxicity due to synergism. In summary, increasing DGLA and concurrently decreasing AA in cells could be a novel approach to controlling the development of AA-dependent cancer. Our study allowed us to directly study the free radical-associated PUFA bioactivity, thus improving our understanding of COX-catalyzed lipid peroxidation in cancer biology.
Detection of Metalloenzymes Employing Fluorescentpolymers and Liposomes
(North Dakota State University, 2012) Dutta, Rinku
In the biological systems, proteins are important constituents. Protein-protein interactions play vital roles in physiological environments and any disruption in these interactions lead to adverse effects. However, designing artificial receptor molecules or scaffolds to imitate or replace these endogenous partners could be an avenue for better drug designing and detection tools creation. We are primarily interested in polymer and liposomal systems to detect two crucial metalloenzymes of the living world. Matrix metalloproteinases are zinc-containing endopeptidases which are required for wound healing, pregnancy and angiogenesis in normal bodily conditions. However, when overexpressed, these cause cancer, arthritis, cardiovascular disorders and fibrosis. Carbonic anhydrases (CAs) are another class of Zn2+ metalloenzymes involved in glaucoma, diabetes, epilepsy and hypertension. Sulphonamide-based inhibitors are prevalent in the market for targeting CAs, but they lack specificities in isozyme-selective inhibition or detection. Usually most of the broad spectrum inhibitors for MMPs have failed the clinical trials due to adverse side effects such as musculoskeletal pain or the inhibition of other non-targeted isozymes. Our strategy was to develop isozyme selective fluorescent water soluble polymers incorporating an active site binding inhibitor for each enzyme class and different charged and uncharged moieties for surface binding with the exposed residues of the isozymes. We have incorporated fluorophores in our polymers which acted as our detection signal generator through fluorescence. For MMPs, one of the optimized polymers was able to detect MMP-9 selectively compared to MMP-7 and -10 (discussed in Chapter 1). This polymer had shown potency in differentiating and subtyping various breast and prostate cancer cell lines from non- cancerous cell lines based on interactions with the secreted MMP-9 from these cell lines (discussed in Chapter II). Chapter III deals with the selective detection of CA II from CA VII and XII even in the complex mixture of biomacromolecules using our synthesized polymers. In Chapter IV, we investigated dye-encapsulated liposomal formulations for detection of catalytically active MMP- 7. The synthesized polymers and liposomes could serve as an alternative detection tool for detection and isozyme selective interactions of these metalloenzymes.
Effect of Dietary Omega-3 and Omega-6 Polyunsaturated Fatty Acids on Alcoholic Liver Disease
(North Dakota State University, 2012) Purwaha, Preeti
PUFAs have been shown to modulate ALD by several mechanisms, including free radical generation from hepatic lipid peroxidation. However, how they modulate lipid peroxidation and generation of bioactive metabolites in ALD is poorly understood and it is still not clear which PUFAs (ω-3 or ω-6) are beneficial or detrimental in ALD. Thus, our objective was to study the effect of ω-3/ω-6 PUFAs on lipid peroxidation and ethanol mediated steatosis and inflammation. Using standard liquid diet (LDC), LDC with fish oil (rich in ω-3) and safflower oil (rich in ω-6), we studied the generation of bioactive metabolites, such as eicosanoids and free radicals generated via lipid peroxidation. In addition, we determined the effect of PUFAs on several inflammatory and fibrotic factors, e.g. gene as well as protein expression, using western blot and RT-PCR, respectively. We also investigated the effect of PUFA diets on novel targets, such as hepatic membrane transporters with potential role in liver inflammation. Our results suggest that ω-3 diet prevented while ω-6 based diets promoted the development of fatty liver and inflammation. ω-3 PUFA reduced AA-peroxidation by lowering hepatic AA concentration and expression of peroxidation enzymes, COX-2 and 5-LOX, resulting in lower generation of pro-inflammatory AA-derived PGs (Series-2), HETEs and free radicals, along with increase in anti-inflammatory EPA and DHA-derived PGs (Series-3). ω-3 diet might also reduce liver inflammation by preventing activation of NF-кB and induction of TNF-α. Rats fed with ω-3 diet showed high protein expression of efflux transporters, MRP-2 and ABCA1, indicating elimination of peroxidation metabolites and triglycerides from the liver and decreased inflammation. In contrast, ω-6 diets led to increase in AA-peroxidation and generation of AA-derived pro-inflammatory metabolites. ω-6 based diets also promoted fatty liver and inflammation by activating NF-кB, inducing TNF-α and downregulation of efflux transporters, MRP-2 and ABCA1. This study not only provides new insights into the effects and possible mechanisms by which ω-3 and ω-6 PUFAs may alter hepatic steatosis and inflammation, but also put forward new targets of research, such as hepatic membrane transporters in relation to liver pathology in ALD.
Inhibition of Nitric Oxide Signaling by Melatonin in Porcine Coronary Arteries
(North Dakota State University, 2012) Shukla, Praveen
This research represents an effort to bridge a critical gap in the understanding of the local effects of melatonin on the coronary circulation. The first objective was designed to identify the specific melatonin receptor mediating the inhibitory effect of melatonin on nitric oxide (NO)-induced relaxation of porcine coronary arteries. Based on current data, one can conclude that the melatonin (MT) type-2 receptor mediates the inhibitory effect of melatonin on coronary arterial relaxation induced by either exogenous or endogenous NO. I have also tested the hypothesis that melatonin inhibits the NO-induced increase in cGMP levels by increasing the degradation of cGMP by cGMP specific phosphodiesterase (PDE). The results suggest that in coronary arteries melatonin acts via MT2-receptors and stimulates the PKG1-dependent PDE5 phosphorylation, resulting in decreased cyclic GMP accumulation in response to NO and impaired NO-induced vasorelaxation. It was further tested whether the impairment of NO-induced relaxation of porcine coronary arteries by melatonin involves inhibition of NO-activated BKCa channel activity in porcine coronary smooth muscle cells. Findings from these studies provide evidence that the impairment of coronary artery relaxation in response to NO by melatonin involves MT2-receptor coupled inhibition of NO-activated BKCa currents. The results of these studies have improved our understanding of the role of melatonin in regulation of local coronary vascular tone. Inasmuch as the NO pathway is a critical regulator of coronary arterial smooth muscle tone, inhibition of this pathway may contribute to the mechanisms by which melatonin influences coronary arterial function in health and disease.
Fluorescent Polymer Based Post-Translational Differentiation and Subtyping of Cancer Cells
(North Dakota State University, 2012) Scott, Michael David
The field of personalized medicine is focused on gathering information at a pre-translational level. Although this information is useful, it is becoming increasingly apparent that post-translational expression is not always consistent with that of the DNA or mRNA. Thus to advance personalized medicine, the development of additional technologies to gather disease state information at post-translational levels are necessary. This dissertation is focused on the advancement of fluorescent polymer technology to monitor at a secretomics level. Secretomics is a subset of proteomics that is focused on secreted proteins/enzymes from the cell. To monitor these secreted proteins, polymers were synthesized from the monomer state, with a polymerizable moiety of 4-vinylbenzoic acid. This compound was conjugated to various amino acids, alcohol derivatives, fluorophores, and metalloproteinase inhibitors. The monomer composition was thus varied to generate a library of polymers capable of forming unique interactions with proteins and enzymes. This polymeric library was screened against recombinant human MMP-7, -9, and -10. The fluorescent emissions data was subsequently evaluated using statistical analysis. This analysis provided information regarding an optimal polymer formulation for MMP isozyme specific interactions. This polymer was subsequently screened against both breast and prostate cancer cell conditioned media. This media were prepared such that the polymers would only interact with secreted proteins/enzymes (i.e. secretomics). Again fluorescence emissions data were evaluated using statistical analysis (linear discriminate analysis [LDA]) to demonstrate the polymer could distinguish between the subtypes, or sub-classification of the cancerous cells. Replacement of the fluorophore with a more hydrophilic pyranine improved the water solubility of the polymer. This polymer was thus screened against both the prostate and breast cancer conditioned media and evaluated using LDA. The results demonstrate an enhanced ability to distinguish and subtype the cancer cells. This dissertation will discuss an alternative mythology for the advancement of post-translational sub-typing with the intent to advance the field of personalized medicine.
Controlled Delivery of Basal Level of Insulin
(North Dakota State University, 2013) Oak, Mayura Arvind
The present study was aimed at developing a delivery system for controlled release of insulin at basal level from chitosan-zinc-insulin complex incorporated into thermosensitive polymer, poly(lactic acid)-poly(ethylene glycol)-poly(lactic acid) (PLA-PEG-PLA). Chitosan-zinc-insulin complex was optimized to restrict the insulin diffusion from the delivery system by complex formation and thereby reducing initial burst release. Polymer concentration, insulin loading, chitosan and Zinc+2 addition were shown to affect the insulin release in vitro. Formulations containing insulin, zinc-insulin, and chitosan-insulin exhibited high initial burst (~7-14%), accompanied with a large secondary burst and incomplete release. Chitosan-zinc-insulin containing formulations showed extended release profiles of insulin for 84-90 days with a significant (P<0.05) reduction in initial burst release and minimal secondary burst. Increasing chitosan amount had no effect (P>0.05) on the initial burst, and release rate. Insulin alone and zinc-insulin containing formulations showed significant (p<0.05) attenuation in secondary and tertiary structure of insulin, as compared to chitosan-zinc-insulin. The complex formation conserved the physical and chemical stability of insulin and protected it from aggregation during release and storage. It also protected insulin from the acidic degradation product of copolymer. The delivery systems were investigated for continuous in vivo insulin delivery at basal level for prolonged period after a single subcutaneous injection. In vivo absorption and bioactivity of insulin released were studied in streptozotocin-induced diabetic rats. Chitosan-zinc-insulin complex significantly (P<0.05) reduced the initial burst release of insulin in comparison to zinc-insulin or insulin alone. The delivery system released insulin for ~70 days in biologically active form with corresponding reduction in blood glucose. Blood glucose levels were comparable to that of control for longer duration, and were significantly (P<0.05) lower than untreated diabetic animals. No significant difference (P>0.05) in blood glucose levels in two consecutive time points until 56-63 days indicated a pharmacodynamic manifestation of continuous release of insulin at steady rate. The delivery systems showed increase in bioavailability of insulin (1.2-2 fold increase in AUC) as compared to zinc-insulin and insulin alone. Insulin released from the delivery systems did not provoke any immune response. The delivery systems were biocompatible in vitro and in vivo and were non-toxic.
Design and Synthesis of Peptide-Based Nanofibers for Imaging and Therapy of Cancer
(North Dakota State University, 2013) Malik, Ruchi
Nanotechnology has been the subject of significant scientific and biomedical development efforts over the past decades. Improvement in biomarker discovery, targeting approaches and conjugation chemistries has led to the development of many novel nanomaterials for individualized therapy. In this thesis, we investigate a new class of nanomaterial called “nanofiber precursor” (NFP). The NFP is composed of multiple self-assembling peptides via electrostatic and non-covalent interactions. Each peptide consisted of β-sheet sequence attached to a methoxypolyethylene glycol (mPEG) via a linker. By conjugating either near infrared fluorophore or therapeutic antibodies, we demonstrate the application of NFP in diagnosis and therapy of cancer respectively. The main objectives of this thesis are: (1) To design and synthesize a near infrared nanofiber for imaging urokinase plasminogen activator (uPA) activity (2) To develop a Herceptin-conjugated nanofiber as multivalent targeted system for increasing therapeutic efficacy of Herceptin, a monoclonal antibody used for breast cancer treatment. We were successful in conjugating near infrared dye NIR664 to the nanofiber as well as Herceptin on the surface of nanofiber. (1) The NIR-NFP conjugate could detect recombinant uPA activity with sensitivity of 3 ng. (2) The Herceptin-conjugated nanofiber (HER-NFP) was more than two fold effective in inhibiting growth of HER-2 positive cells. In the second half of the thesis, we have also investigated tumorigenic role of 15-LOX-1, a lipid peroxidizing enzyme in prostate cancer. The aim of this study was: (3) To investigate the role of 15-lipoxygenase-1 (15-LOX-1) in upregulation of uPA in PC-3 prostate cancer cells. As a whole, the research presented in this thesis is aimed at designing new strategies and understanding molecular mechanisms that lead to prevention and treatment of cancer.
Angiotensin (1-7) Attenuates the Chronotropic Response to Angiotensin II via Stimulation of PTEN in Spontaneously Hypertensive Rat Brain
(North Dakota State University, 2013) Modgil, Amit
The pathogenesis of hypertension and its mode of progression are complex, multifactorial and incompletely understood. Several studies have focused on the beneficial effects of peripheral Ang (1-7) in the regulation of cardiovascular functions, showing the counter-regulatory effects of Ang (1-7) against the actions of Ang II in the periphery. However, its actions in the central nervous system are not completely understood. In the present study, our main goal was to determine the central action of Ang (1-7) and its interaction with Ang II in the blood pressure control. Previous studies reported that Ang II produces a greater degree of activation of neuronal cells from brainstem/hypothalamus cultures of SHR versus WKY rats. Our present findings showed that this enhanced action of Ang II was attenuated in co-presence of either Ang (1-7) or PI3-kinase inhibitor. These counter-regulatory effects of Ang (1-7) on Ang II action in SHR neurons were abolished by co-treatment with either A-779, a Mas-R antagonist, or bisperoxovanadium (BPV), a PTEN inhibitor. In addition, incubation of WKY and SHR neurons with Ang (1-7) significantly increased PTEN activity. Chronic treatment with Ang (1-7) or chronic inhibition of PI3K using lentiviral vector significantly abolished the enhanced chronotroic response to Ang II in SHR neurons and significantly enhanced PTEN protein and mRNA expression levels in both WKY and SHR neuronal cultures. To further check the functional implications of our in vitro data, we further studied the interaction between Ang II and Ang (1-7) in the central control of cardiovascular functions. RVLM microinjection of Ang (1-7) or LY-294002 alone did not alter MAP, but reduced the pressor response to Ang II in SHR. Moreover, in compliance with our in vitro data, the inhibitory effect of Ang (1-7) on the pressor response to Ang II in SHR was abolished when co-administered together with A-779 or BPV. The data demonstrated that Ang (1-7) induce PTEN activity and expression via Mas-R, and depresses PI3-kinase-PKB/Akt signal transduction pathway, which lead to the counter-regulatory effect of Ang (1-7) on Ang II induced chronotropic and pressor effect on neuronal activity and cardiovascular functions including MAP and HR in SHR.
Mir-627 Mediates the Epignetic Mechanisms of Vitamin D in Suppression of Colon Cancer Growth
(North Dakota State University, 2013) Padi, Sathish Kumar Reddy
Low circulating vitamin D levels have been linked to increased risks of cancer by epidemiologic studies, and preclinical research has demonstrated clear antitumor activities of vitamin D against various types of cancers. Calcitriol (1a, 25(OH)2 D3), the active form of vitamin D, has been shown to inhibit proliferation, induce differentiation, and activate apoptosis in colon cancer cells. However, the mechanism of calcitriol's antitumor action remains poorly defined and the hypercalcemic side effect of vitamin D limits its use as an anti-cancer agent. To understand its mechanism of action in tumor suppression, we investigated the effects of calcitriol on microRNA expression in colon cancer cells. We identified miR-627 as the microRNA whose expression was most significantly stimulated by calcitriol. Furthermore, JMJD1A (jumonji domain containing 1A), the gene encoding a histone demethylase, was identified as the target of miR-627. It has been reported that JMJD1A is upregulated in colon cancer during hypoxia. Increased JMJD1A expression decreases the histone methylation on the promoters of target genes such as adrenomedullin (ADM) and the growth and differentiation factor 15 (GDF15) and increases their expression, which promotes tumor growth. By downregulating JMJD1A, miR-627 mediated the effects of calcitriol to increase histone H3K9 methylation and to suppress the expression of growth-promoting genes, such as GDF15. Calcitriol also induced miR-627 and downregulated JMJD1A in colon cancer xenografts in nude mice. Calcitriol significantly suppressed the growth of colon tumors in nude mice, whereas colon tumor xenografts with stable JMJD1A-UTR expression showed resistance to the Calcitriol treatment, confirming the critical role of miR-627 in Calcitriol induced tumor suppression. Overexpression of miR-627 decreased JMJD1A and suppressed colon cancer growth both in vitro and in vivo. In addition, we found that miR-627 expression was decreased in human colon adenocarcinomas compared with controls. These results suggest that, miR-627 is a major epigenetic regulator in vitamin D induced growth inhibition. MiR-627 and JMJD1A may serve as potential targets to exploit the antitumor activity of vitamin D without eliciting its hypercalcemic side effect.
In Vitro Detection of Disease Biomarkers and Drug Contaminants
(North Dakota State University, 2013) Nyren-Erickson, Erin Kathryn
In recent years the rising cost and increased regulation within the U.S. healthcare system have caused medical laboratory tests to become more costly and more frequently required. As a result, insurance premiums are rising, and small independent laboratories are threatened with closure as their already narrow margins dwindle. Concurrently, there have been several incidents of contaminants and impurities in pharmaceutical drugs causing hundreds of deaths and thousands of illnesses. These challenges substantiate the need for simple and cost-effective screening tests for the presence of disease biomarkers, as well as for contaminants and impurities present in pharmaceutical drugs. The following disquisition reports three independent studies, each with the development of simple screening tools as its objective. Paper 1 reports the use of fluorescent lipid nanoparticles (liposomes) to detect changes in the species and concentrations of glycosaminoglycans (GAGs) in solution. We conclude that the emission intensity from the present fluorophores changes in response to increasing concentrations of GAGs, and can distinguish between serum from a healthy patient and serum having the same GAG concentrations as an Alzheimer's disease patient (simulated). Paper 2 reports the use of lipid nanoparticles to detect dangerous over-sulfated contaminants in pharmaceutical heparin. We report that liposomes in the presence of heparin or over-sulfated contaminants and Mg2+ ions form aggregates, and the size and zeta potential of these aggregates is dependent on the heparin/contaminant present. Further, the variation in aggregate zeta potential varies significantly upon heparin contamination, and may be used to detect 0.5% contaminant by weight. Paper 3 reports a clinical study to validate the presence of ADAM 12 (a disintegrin and metalloproteinase) enzyme in urine as a biomarker for breast cancer detection and diagnosis, as well as to monitor the effects of tumor removal on the urinary levels of this enzyme. We find no significant differences between recently diagnosed cancer patients (having undergone no treatment for cancer) and age-matched controls having no cancer present. Significant increases in urinary ADAM 12 only occur following surgery. Overall, we conclude that it is unlikely that a screen for urinary ADAM 12 will be useful for the diagnosis of breast cancer.
Development of New Antibody Based Theranostic Agents Targeting the Receptor for Advanced Glycation End-Product (Rage)
(North Dakota State University, 2013) Jyoti, Faidat
The Receptor for Advanced Glycation End products (RAGE) interacts with several classes of structurally unrelated ligands. The activation of RAGE by its ligands results in the cellular activation of several kinases and transcription factors including mitogen activated protein kinases (MAPKs) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) resulting in sustained inflammation, which is involved in pathologies such as diabetes, cancer, Alzheimer's disease, multiple sclerosis and other diseases associated with chronic inflammation. Current mouse models of human disease have shown that RAGE activity can be efficiently suppressed using either soluble RAGE (sRAGE) or anti-RAGE antibodies as inhibitors. Our goal was to generate new monoclonal antibodies against RAGE that can serve as diagnostic as wells as therapeutic tools in RAGE related pathologies. The chapters in this dissertation are a complete documentation of the development of these anti-RAGE antibodies. Additionally, an introductory review of antibodies, which includes structure and function, types of antibodies and production and basic understanding of RAGE and its ligands, has been provided to facilitate the understanding of the chapters. The first chapter details the development and characterization of anti-RAGE antibodies produced from hybridoma. The next chapter explores the effects of the generated antibodies to mammalian cells in in vitro settings and the final chapter applies the generated antibodies in vivo. During the course of this work, the antibodies developed showed binding to RAGE at nano-molar affinities which are comparable to the affinities of current antibodies used for therapeutic purposes, diagnostic and research purposes. We were also able to delineate that the possible mechanism of action of the antibodies is by preventing binding to RAGE. Lastly, we observed that one of the generated antibodies was able to reduce tumor growth in vivo in a melanoma xenograft mouse model.
Synthesis and Evaluation of Cationic Nanomicelles for In Vitro and In Vivo Gene Delivery
(North Dakota State University, 2013) Mandke, Rhishikesh Subhash
The goal of proposed study was to contribute towards the development of a nano size, high efficiency and low toxicity non-viral polymeric vector for gene delivery in vitro and in vivo. A series of fatty acid grafted low-molecular-weight chitosan (N-acyl LMWCs) were synthesized, purified and characterized for their physicochemical properties using various analytical techniques such as infrared spectroscopy, elemental analysis and dynamic light scattering. The formulation parameters including pH, sonication duration, and filtration altered the physicochemical characteristics of N-acyl LMWC nanomicelles. The acyl chain length and degree of unsaturation in fatty acids also had an impact on the physicochemical properties and the transfection efficiency of nanomicelles. N-acyl LMWC nanomicelles showed efficient in vitro transfection as visualized and quantified using a reporter plasmid (encoding green fluorescent protein), and therapeutic plasmids (encoding for interleukin-4 and interleukin-10), respectively. The in vitro transfection efficiencies of N-acyl LMWCs with 18:1 and 18:2 grafts (oleic and linoleic acids) were comparable with FuGENE® HD (marketed non-viral vector) but were ~8-fold and 35-fold higher as compared to LMWC and naked DNA, respectively. The in vivo transfection efficiency of N-acyl LMWC to deliver plasmids individually encoding IL-4 and IL-10 as well as a bicistronic plasmid encoding both IL-4 and IL-10 was studied in a multiple, low-dose streptozotocin induced diabetic mouse model. The transfection efficiency of pDNA/N-acyl LMWC polyplexes injected via intramuscular route showed significant improvement (p<0.05) over passive (naked DNA) or positive (FuGENE HD) controls. Additionally, a sustained and efficient expression of IL-4 and IL-10 was observed, accompanied by a reduction in interferon-gamma (INF-γ), and tumor necrosis factor-alpha (TNF-α) levels. The pancreas of pDNA/N-acyl LMWC polyplex treated animals exhibited protection from streptozotocin-induced insulitis and the delivery systems were biocompatible. Histological studies revealed that there were no signs of chronic inflammation at the injection site. The bicistronic plasmid exhibited significantly (p<0.05) greater expression of IL-4 and IL-10, and demonstrated the feasibility of bicistronic IL-4/IL-10 plasmid/N-acyl LMWC nanomicelles-based polyplexes as an efficient and biocompatible system for the prevention of autoimmune diabetes.
Design and Evaluation of Polymeric Nanomaterials for In Vitro and In Vivo Imaging Applications
(North Dakota State University, 2014) Wagh, Anil V.
One of the most versatile and safe material used in medicine is polymer-based nanomaterials. This dissertation describes the use of several formulations of polymeric nanomaterials for in vitro and in vivo optical imaging applications. In the first phase of this work, the particles assembled from diblock copolymers of poly(D,L-lactic-co-glycolic acid) and polyethylene glycol were used as a carrier for diagnostic agents. In chapter 2, the polymeric nanoparticles with a large Stokes shift of >100 nm were employed for in vivo imaging. The large Stokes shift was achieved through fluorescence resonance energy transfer (FRET) by encapsulating the donor (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine) and acceptor (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine) fluorophores inside a single nanoparticle. These nanoparticles were then systematically explored to optimize the fluorophore loading and the maximum energy transfer efficiency. The animal studies further demonstrated that these nanoparticles could have far-reaching applications for in vivo imaging. In chapter 3, we further extended the study by doping the combinations of four different fluorophores, DiO, Dil, DiD, and DiR to synthesize particles that exhibited distinct emission signatures ranging from the visible to near-infrared wavelength region. This work presents first instance of nanoparticles encapsulated with four different energy transfer fluorophores inside a single particle. The optimized multicolor nanoparticles could simultaneously emit fluorescence at three different wavelengths (at 570, 669, and 779 nm) upon a single excitation (at 485 nm). Furthermore, particles with single, double, and triple emissions could be synthesized by changing the combination and doping ratio of the fluorophores. We further demonstrated that this technology could be applied to multicolor and multiplex imaging. Various physiological mechanisms are responsible for nanomaterial interaction and clearance from the blood circulation. The objective of chapter 4 was to investigate the biocompatibility, pharmacokinetics, and biodistribution of peptide-based nanofiber (NFP). In vitro studies suggested that NFP is non-toxic, hemocompatible and only showed a minimum uptake by the isolated macrophages. Upon systemic injection into mice, NFP could be delivered to the tumor in a short period of time and also eliminated rapidly by renal clearance. Overall, our results suggested that NFP is a biocompatible, safe, and effective carrier for tumoral delivery.
Dual Modified Liposomes for Drug and Gene Delivery to Brain
(North Dakota State University, 2014) Sharma, Gitanjali
The overall goal of our research was to design a vector for efficient delivery of therapeutic genes/drugs to brain. Specifically, this research work was focused on designing PEGylated liposomes surface modified with the receptor targeting protein, transferrin and cell penetrating peptides (CPPs) for targeting and improving the delivery of desired therapeutic agent to brain. Various CPPs including poly-L-arginine, TAT, Penetratin and Mastoparan were investigated for their influence on transport of transferrin receptor targeted liposomes across brain endothelial cells. The dual-modified liposomes were synthesized using thin film hydration and post-insertion technique. The biocompatibility of the liposomes was evaluated at increasing concentrations to obtain an optimum value for safe and effective delivery of drugs or genes. The liposomes showed excellent cellular, blood and tissue compatibility at the optimized concentration. In addition, the combination of targeting ligand transferrin and CPPs resulted in considerable translocation of the therapeutic agent across cellular and brain endothelial barriers both in vitro and in vivo. Among different Tf-CPP liposomes, the Tf-Penetratin liposomes showed maximum translocation of the drug across the brain endothelial barrier (approximately 15% across in vitro and 4% across in vivo BBB) and efficient cellular transport of the encapsulated drug (approximately 90-98%) in various cell lines. In addition, Tf-poly-L-arginine and Tf-Penetratin liposomes showed improved transfection efficiencies in various cell lines. The Tf-Penetratin and Tf-TAT liposomes demonstrated excellent cellular biocompatibility and no hemolytic activity upto 200nM phospholipid concentration. In vivo efficacy of the liposomes was evaluated by performing biodistribution studies in in adult Sprague Dawley rats. The liposomes were intended for delivery of small molecule drug, doxorubicin and pDNA to brain. The dual modified liposomes showed significantly (p<0.05) higher transport of encapsulated agents in rat brain as compared to single ligand (Tf) or plain liposomes. Histological examination of the tissues, from various organs, did not show any signs of toxicity including necrosis, inflammation, fibrosis etc. The study underlines the potential of bifunctional liposomes as high-efficiency and low-toxicity gene delivery system for the treatment of central nervous system disorders.
Development and Characterization of Multifunctional Nanoparticles for Drug Delivery to Cancer Cells
(North Dakota State University, 2014) Nahire, Rahul
Lipid and polymeric nanoparticles, although proven to be effective drug delivery systems compared to free drugs, have shown considerable limitations pertaining to their uptake and release at tumor sites. Spatial and temporal control over the delivery of anticancer drugs has always been challenge to drug delivery scientists. Here, we have developed and characterized multifunctional nanoparticles (liposomes and polymersomes) which are targeted specifically to cancer cells, and release their contents with tumor specific internal triggers. To enable these nanoparticles to be tracked in blood circulation, we have imparted them with echogenic characteristic. Echogenicity of nanoparticles is evaluated using ultrasound scattering and imaging experiments. Nanoparticles demonstrated effective release with internal triggers such as elevated levels of MMP-9 enzyme found in the extracellular matrix of tumor cells, decreased pH of lysosome, and differential concentration of reducing agents in cytosol of cancer cells. We have also successfully demonstrated the sensitivity of these particles towards ultrasound to further enhance the release with internal triggers. To ensure the selective uptake by folate receptor- overexpressing cancer cells, we decorated these nanoparticles with folic acid on their surface. Fluorescence microscopic images showed significantly higher uptake of folate-targeted nanoparticles by MCF-7 (breast cancer) and PANC-1 (pancreatic cancer) cells compared to particles without any targeting ligand on their surface. To demonstrate the effectiveness of these nanoparticles to carry the drugs inside and kill cancer cells, we encapsulated doxorubicin and/or gemcitabine employing the pH gradient method. Drug loaded nanoparticles showed significantly higher killing of the cancer cells compared to their non-targeted counterparts and free drugs. With further development, these nanoparticles certainly have potential to be used as a multifunctional nanocarriers for image guided, targeted delivery of anticancer drugs.
Rage (Receptor for Advanced Glycation End Products) in Melanoma Progression
(North Dakota State University, 2014) Meghnani, Varsha
The Receptor for Advanced Glycation End Products (RAGE) and its ligands are expressed in multiple cancer types and are implicated in cancer progression. Our lab has previously reported higher transcript levels of RAGE and S100B in advanced staged melanoma patients. The contribution of RAGE in the pathophysiology of melanoma has not been well studied. Based on previous reports, we hypothesized that RAGE, when over-expressed in melanoma cells, promotes melanoma progression. To study the pathogenic role of RAGE in melanoma, a primary melanoma cell line, WM115, was selected and stably transfected with full length RAGE (FL_RAGE) to generate a model cell line over-expressing RAGE (WM115_RAGE). WM266, a sister cell line of WM115, originated from a metastatic tumor of the same patient was used as a metastatic control cell line in the study. After assessing the expression levels of RAGE in the transfected cells, the influence of RAGE on their cellular properties was examined. An enhanced motility but suppressed proliferation of WM115 cells was found after RAGE over-expression, these properties could be reversed upon suppression of RAGE in these cells. We next explored the mechanisms of RAGE induced changes in cell proliferation and migration in WM115_RAGE cells and found a significant upregulation in S100B protein in the WM115_RAGE melanoma cells compared to the MOCK cells. However, S100B suppression produced no effect on WM115_RAGE cells motility. Furthermore, expression of tumor suppressor p53 protein, which is one of the target proteins of S100B, was found to be significantly reduced in WM115 cells after RAGE over-expression. We also investigated the effect of RAGE over-expression on melanoma tumor growth and deciphered the downstream signaling involved. We found a significantly larger tumor growth rate of the WM115_RAGE cells compared to the control cells. S100B, S100A4, S100A6 and S100A10 proteins that are relevant in melanoma pathophysiology, were found to be upregulated in WM115_RAGE tumors compared to MOCK tumors. Moreover, enhanced AKT and ERK signal activation was observed in WM115_RAGE tumors as compared to MOCK tumors. Finally, anti-RAGE antibody treatment significantly suppressed tumor growth, which could be further enhanced by combining the antibody with the chemotherapeutic drug dacarbazine.
Evaluation of Hydrophobically Modified Low Molecular Weight Chitosan as a Potential Nonviral Vector for DND Vaccine Delivery
(North Dakota State University, 2014) Layek, Buddhadev
Gene therapy has great potential in disease prevention and treatment. The purpose of the proposed research was to advance the development of safe and effective nonviral polymeric vectors for targeted gene therapy and DNA vaccine delivery. A series of hydrophobically modified chitosan derivatives with increasing degrees of chain length, substitution, and hydrophobicity was synthesized via carbodiimide mediated coupling reaction. The chemical structure of the polymers was determined using proton nuclear magnetic resonance (1H NMR), fourier transform infrared (FTIR) spectroscopy, and elemental analysis. These polymers form micellar structures in aqueous environment and effectively condense plasmid DNA (pDNA) into nanoscale polyplexes. The acyl chain length, degree of substitution, and hydrophobicity of the substituent had great impact on the particle size, pDNA binding strength, in vitro pDNA release profile, cellular uptake, and in vitro gene transfection efficiency of the polymer/pDNA polyplexes. The hexanoic acid grafted chitosan [NAC-6(15)] and L-phenylalanine grafted chitosan (AGC-F) with a 15% degree of amino substitution demonstrated significantly (p < 0.05) higher gene transfection in HEK 293 cells, and their transfection efficiency surpassed the transfection capacity of FuGENE HD. The NAC-6(15) and AGC-F polymers were mannosylated to provide selective antigen presenting cell (APC) targeting, thereby facilitating cellular uptake to ultimately improve the overall immune response to the DNA vaccine. The chemical composition of the mannosylated copolymers was analyzed by 1H NMR spectroscopy. These polymers efficiently condense pDNA into nanosized polyplexes with net positive surface charges. The resultant polyplexes demonstrated excellent protection of the condensed pDNA from enzymatic degradation by deoxyribonuclease (DNase). The synthesized mannosylated polymers exhibited 7-fold greater cellular uptake than chitosan in RAW 264.7 cells, which express mannose receptors, mainly via the receptor-mediated endocytosis without affecting biocompatibility. The in vitro transfection efficiencies of mannosylated polymer/pDNA polyplexes were significantly (p < 0.05) higher than other polymers and FuGENE HD. An in vivo study in Balb/c mice using hepatitis B surface antigen encoding pDNA as a model DNA vaccine reflected good efficacy and biocompatibility of the delivery system. Therefore, hydrophobically modified mannosylated chitosan derivatives have the potential to be a safe and efficient APC targeting gene carrier for DNA vaccine delivery.
Identification and Characterization of Binding Target Proteins of Cancer Stem Cell Inhibitor Salinomycin in Human Neuroblastoma
(North Dakota State University, 2015) Zhou, Shuang
Salinomycin, a widely used anti-coccidial agent, was recently identified as a cancer stem cell (CSC) inhibitor from a library of 16,000 natural and commercial chemical compounds based on its highly selective inhibitory effect on breast CSCs, with more than 100-fold greater potency than paclitaxel. Salinomycin also exhibits cytotoxic effects on other types of cancer cells and CSCs and overcomes drug resistance. However, the exact mechanism of salinomycin, especially its direct binding target(s), and its effects on Neuroblastoma (NB) are yet not known. NB is a common solid tumor and a leading cause of mortality in children. Currently, 35% of patients with NB remain incurable. In addition, the majority survivors of NB suffer from long-term side effects of current therapies and are at risk for disease relapse or getting a second, different cancer. More effective therapies are pressingly needed. Since the existence of CSCs in human NB cell lines and NB tumors has been well documented, and has been closely associated with chemoresistance or tumor relapse, therapeutic targeting of NB CSCs may be a critical novel approach for NB therapy. Aiming to improve NB therapy, we examined the efficacy and mechanism of salinomycin in human NB cells. Our study showed that salinomycin markedly inhibits NB cell proliferation and tumorsphere formation. Treatment of salinomycin induced G2 cell cycle arrest with an up-regulation of Cyclin A and a down-regulation of p21 protein levels. We further identified Transcription intermediary factor 1-beta (TIF1β) and Nucleolin (NCL) as novel binding targets of salinomycin by using comprehensive methods, including chemical proteomics and functional genomics. We demonstrate that salinomycin induced phosphor-TIF1β mediated down-regulation of p53 and significantly suppressed the expression of CD34, a CSC marker, via disrupting the interaction of NCL with the CD34 promoter. Furthermore, by analyzing tumor samples data from a cohort of 498 NB patients, we found that elevated levels of TIF1β and NCL in NB are associated with a poor outcome. These results provide a therapeutic rationale for evaluating of both salinomycin and its binding proteins, TIF1β and NCL, in NB.
Epigenetic Regulation of Apoptosis in Prostate Cancer
(North Dakota State University, 2015) Zhang, Qunshu
Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 and suppresses gene expression by catalyzing histone H3 methylation on lysine 27. EZH2 is overexpressed in metastatic prostate cancer and has been shown to promote cell proliferation and metastasis. Here we show that EZH2 also suppresses prostate cancer apoptosis by coordinating the epigenetic silencing of two pro-apoptotic microRNAs, miR-205 and miR-31. We previously reported that miR-205 is silenced in prostate cancer through promoter methylation. In this study, we found that EZH2 suppresses miR-31 expression by trimethylation of H3K27 on the miR-31 promoter. SiRNA knockdown of EZH2 increased miR-31 expression and decreased the anti-apoptotic protein E2F6 (a target of miR-31), resulting in the sensitization of prostate cancer cells to docetaxel-induced apoptosis and vice versa. We further demonstrated that miR-205 silencing is linked to miR-31 silencing through EZH2. Suppression of miR-205 caused an increase of EZH2 protein, which in turn inhibited miR-31 expression and vice versa. Thus, EZH2 integrates the epigenetic silencing of miR-205 and miR-31 to confer resistance to chemotherapy-induced apoptosis. Besides the histone modification by histone methyltransferases (HMTs) such as EZH2, histone deacetylases (HDACs) offer another mechanism to epigenetically regulate gene expressions in cancer. The class I selective inhibitor of HDACs, mocetinostat, has promising antitumor activities in both preclinical studies and the clinical trials. To understand how mocetinostat induces apoptosis in prostate cancer cells, we examined the effects of mocetinostat on miR-31. We found that miR-31 was significantly upregulated by mocetinostat in prostate cancer cells. E2F6 was decreased by mocetinostat treatment. Mocetinostat also increased the expression of pro-apoptotic protein Bad and activated caspase-3 and caspase-9. SiRNA iv knockdown of E2F6 sensitized cancer cells to mocetinostat-induced apoptosis. Importantly, we found the same results in the primary prostate cancer stem cells. Thus, activation of miR-31 and downregulation of E2F6 contribute to mocetinostat-induced apoptosis in prostate cancer. In summary, the epigenetic silencing of miR-31 confers a resistance mechanism for chemotherapy-induced apoptosis in prostate cancer cells. Using mocetinostat to activate miR-31 expression is a novel strategy to overcome resistance to apoptosis and improve response to therapy.
Interactions of the Receptor for Advanced Glycation End Products (Rage) with Advanced Glycation End Products (AGEs) and S100B
(North Dakota State University, 2016) Indurthi, Venkata
RAGE is a multi-ligand pattern recognition receptor. RAGE can bind several damage associated molecular pattern proteins. RAGE- ligand interaction is pathophysiologically relevant to several major diseases including diabetes and certain cancers. RAGE inhibition has been reported to reduce morbidity in these disease states. However, to design better RAGE inhibitors it is necessary to understand the structural basis behind the RAGE-ligand interaction and currently this is not well understood. This thesis focuses on understanding the interaction of RAGE with two of its ligands; AGEs and S100B. AGEs are highly heterogeneous and are formed as a result of non-enzymatic glycation. A panel of AGEs were characterized in terms of their side chain modifications, thermal stability, secondary structure, aggregation and surface charge. These glycation induced changes were then correlated to RAGE binding. Building on these results the role of AGE-RAGE interaction in pancreatic cancer cell proliferation and migration was determined. Ribose modified BSA induced ROS formation, which then triggered NF-κB upregulation via RAGE induced ROS signaling. Ribose BSA increased pancreatic cell proliferation and migration. Anti-RAGE antibodies and RAGE inhibitors prevented AGE induced cellular effects. The role of ribose modified BSA was also determined in macrophage activation and pro-inflammatory cytokine release. Rapid internalization was observed of the ribose-BSA and confocal imaging revealed the internalization of the AGE compound into the lysosomes which lead to the ROS production, NF-κB activation and pro-inflammatory cytokine release in a RAGE independent signaling mechanism. Finally, the role of tryptophan residues of the V domain in domain stability and S100B binding was determined. We have generated single, double and triple tryptophan mutants of the V domain by site directed mutagenesis. The effect of Trp residues in the domain stability could not elucidated as no change was observed in the secondary structure of the mutants when compared to the wild type suggesting the plasticity of the V-domain. The fluorescence emission and life time properties of each Trp residue was determined. Our binding assays of the Trp Ala mutants indicate tighter binding of the S100B to the mutants. The S100-RAGE peptide structures suggest multi modal interaction of S100B-RAGE interaction.

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