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Item The Role of the Gut Microbiota in Sustained Weight Loss Following Roux-en-Y Gastric Bypass Surgery(North Dakota State University, 2018) Fouladi, FarnazRoux-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.Item Smart, Biocompatible Polymersomes for Targeted Delivery of Therapeutic Agents to Cancerous Tumors(North Dakota State University, 2017) Anajafi Marzijarani, TayebehChemotherapeutics are the major treatment options for cancer. Although we cannot underestimate the importance of the chemotherapeutic drugs, their systemic toxicity is an important limiting factor for their use. Therefore, altering the biodistribution of the therapeutics can be an important step in treating the cancer patients. Thus, there is a growing interest in developing smart, targeted, stimuli responsive, drug delivery vehicles employing nanotechnalogy. The vehicles are often engineered to deliver the theranostics to the tumor microenvironment (extracellular matrix) or intracellular environment (e.g. cytosol, nucleus, mitochondria). The physiochemical changes in the cancerous tissues (e.g. leaky vasculature, proteins overexpression on the cell surface, overexpression of proteolytic enzymes, increased reducing agents concentration, decreased pH, and hypoxia) offer tremendous opportunities for selectively targeting the malignancies. Polymersomes are robust polymeric vesicles which have shown promising drug delivery capabilities. Both hydrophilic and hydrophobic compounds can be loaded to the vesicles. The polymersome's building blocks can be chemically manipulated to provide them with the optimum release profile. Furthermore, the surface of the polymersomes can be decorated with targeting moieties such as peptides, antibodies, or small molecules. We have developed smart, targeted, stimuli responsive polymersomes to combat pancreatic and prostate cancers.Item Dual-Functionalized Liposomes for Gene Delivery to Brain to Prevent and Treat Alzheimer’s Disease(North Dakota State University, 2019) dos Santos Rodrigues, BrunaAlzheimer’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.Item A Nano-Sized Approach to Exploiting the Pancreatic Tumor Microenvironment(North Dakota State University, 2020) Confeld, Matthew IanMaking 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.Item 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, YanCyclooxygenase (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.Item Controlled Delivery of Basal Level of Insulin(North Dakota State University, 2013) Oak, Mayura ArvindThe 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.Item Design and Synthesis of Peptide-Based Nanofibers for Imaging and Therapy of Cancer(North Dakota State University, 2013) Malik, RuchiNanotechnology 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.Item Ang II-Induced Cardiac Remodeling: Role of PI3-Kinase-Dependent Autophagy(North Dakota State University, 2018) Zhong, TiechengHeart failure (HF) is a pathological state indicating insufficient blood supply to the peripheral tissues from the heart. The pathophysiology of HF is multifactorial like cardiac remodeling including cardiac hypertrophy, perivascular fibrosis and apoptosis to compensate for the heart’s inability to pump enough blood. Cardiac hypertrophy is initially adaptive to hemodynamic overload; however, it chronically contributes to heart failure and sudden cardiac death. The extracellular regulatory factors and intracellular signaling pathways involved in the cardiac remodeling are not yet fully clear. PI3-kinase is an important intracellular kinase in organ size control. Cardiac overexpression of Class I PI3-kinase caused heart enlargement in transgenic mice. Autophagy as a dynamic process involving the degradation of damaged mitochondria prevents ROS overproduction which leads to the cardiac remodeling. Therefore, our aim was to study the relationship between PI3-kinases and Ang II-induced cardiac remodeling via an autophagy-dependent mechanism. Ang II significantly increased autophagy with two distinctive phases: an increasing phase at low doses and a decreasing phase at high doses in cardiomyocytes. The Ang II-induced autophagic depression was attenuated by a Class I PI3-kinase inhibitor and potentiated by Class III PI3-kinase inhibitor. Besides, Ang II-induced cardiac hypertrophy and mitochondria ROS generation were attenuated via blockade of Class I PI3-kinase or mTOR. To further validate our in vitro data, we studied the role of Class I PI3-kinase in Ang II-induced cardiac remodeling in vivo. We successfully transferred Lv-DNp85 (Class I PI3-kinase blockade) and Lv-GFP (control) into adult rat hearts and found that cardiac transfer of Lv-DNp85 did not alter Ang II-induced pressor effect, but attenuated Ang II-induced cardiac hypertrophy, perivascular fibrosis and cardiac dysfunction. Ang II-induced cardiac remodeling was associated with impaired autophagy and mitochondrial ROS overproduction, which were significantly attenuated by Lv-DNp85-induced blockade of Class I PI3-kinase. Taken together, these data suggest that Class I PI3-kinase is involved in Ang II-induced impairment of autophagy via Akt/mTOR pathway, leading to mitochondrial ROS overproduction and cardiac remodeling. These results are not only highly significant from a pathophysiological perspective, but also have important pharmacological implications in the control of cardiac hypertrophy to prevent decompensation and failure in cardiac function.Item 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 RaquibulOsteomyelitis 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.Item Detection of Metalloenzymes Employing Fluorescentpolymers and Liposomes(North Dakota State University, 2012) Dutta, RinkuIn 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.