Pharmacy Doctoral Work

Permanent URI for this collectionhdl:10365/32397

Browse

Now showing 1 - 20 of 51

Ang II-Induced Cardiac Remodeling: Role of PI3-Kinase-Dependent Autophagy
(North Dakota State University, 2018) Zhong, Tiecheng
Heart 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.
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.
Angiotensin-(1-7): A Target for Stem Cell Mobilopathy and Vascular Repair in Diabetes
(North Dakota State University, 2017) Vasam, Goutham
Bone marrow stem/progenitor cells (BMPCs) accelerate vascular repair by re-endothelialization and revascularization of ischemic areas. Diabetes causes impairment of BMPC mobilization, a.k.a. stem cell mobilopathy, and reparative functions, which have now been considered as a major contributing factor for the development of macro and microvascular complications and end-organ damage. Therefore, autologous cell therapies for the treatment of diabetic vascular complications are currently not possible. In this study, I tested the effects of Angiotensin (Ang)-(1-7), a heptapeptide member of the protective arm of renin-angiotensin system, on mobilization of BMPCs and their ischemic vascular repair functions that are impaired in diabetes. Streptozotocin-induced diabetic or db/db mice were used. Circulating and bone marrow Lineage- Sca1+ c-Kit+ (LSK) cells were decreased in diabetes, which was normalized by Ang-(1-7). Ang-(1-7) specifically increases Rho-kinase (ROCK) activity in diabetic bone marrow (BM) LSK cells, and fasudil, a ROCK inhibitor, prevented the beneficial effects of Ang-(1-7). BM Slit3 levels were increased by Ang-(1-7), which might have activated ROCK in LSK cells and sensitized for stromal-derived factor-1 (SDF)-induced migration. In relation to ischemia, diabetes prevented LSK cell mobilization and blood flow recovery, which were reversed by Ang-(1-7). Ang-(1-7), in combination with G-CSF or plerixafor reversed the stem cell mobilopathy in diabetes. These beneficial effects of Ang-(1-7) were blunted in Mas receptor knockout (MasR-KO) mice. These results suggest that MasR is a promising target for the treatment of diabetic bone marrow mobilopathy and vascular disease. Overall, this study provided strong preclinical evidence, supporting Ang-(1-7) as a promising molecule for the treatment of diabetic stem cell mobilopathy and vascular disease.
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.
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.
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.
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.
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 Development of Tumor Microenvironment Responsive PEGylated Nanoparticles for Drug Delivery to Cancerous Solid Tumors
(North Dakota State University, 2016) Kulkarni, Prajakta
Rapid growth of cancerous cells creates a biochemically distinct microenvironment in solid tumors. Leaky vasculature, lower pH, increased levels of proteolytic enzymes, hypoxia serve as hallmarks of tumor tissues. These changes in the tumor microenvironment present with opportunities to deliver drug at the targeted tumor tissues using stimuli responsive PEGylated nanoparticles. Stimuli responsive PEGylated nanoparticles extravasate into the tumor tissues through leaky vasculature developed at the tumor site. In the tumor tissue they undergo changes in the physico-chemical properties of the nanoparticle leading to stimuli responsive release of the entrapped chemotherapeutic/imaging agents. Clinical use of PEGylated liposomal doxorubicin formulation has encouraged multiple studies to improve the efficacy of the treatment and reduce side effects of chemotherapy. Liposomes and polymersomes are nanoparticles which form a lipid or polymeric bilayer allowing entrapment of hydrophilic molecules at the core and lipophilic molecules in the bilayer. These chemically engineered drug carriers allow targeting and drug delivery preferentially at the pathologically affected tissues. Stimuli responsive liposomes and polymersomes hold tremendous potential for drug delivery to solid tumors. We have prepared tumor microenvironment responsive PEGylated liposomes and polymersomes for efficient drug delivery to pancreatic cancer cells.
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.
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.
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.
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.
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.
Development of a Novel Omega-6 Fatty Acid Based Treatment Strategy for Colon Cancer by Knocking Down Delta-5-Desaturase and Exploiting High COX-2 Levels in Cancer CellsTumors
(North Dakota State University, 2017) Xu, Yi
Colon cancer is the third most commonly diagnosed cancer in the world. Research showed that arachidonic acid, a downstream ω-6 fatty acid (ω-6), plays a role in colon cancer development by producing deleterious metabolites from its COX-2 catalyzed peroxidation. On the other hand, dihomo-γ-linolenic acid (DGLA), the immediate precursor of arachidonic acid, may represent an exceptional ω-6 associated with anti-cancer activities. However, the mechanism of DGLA’s anti-cancer effect still remains unclear, and the rapid conversion of DGLA to arachidonic acid in human body by delta-5 desaturase (D5D) greatly restricts DGLA’s availability. Recent work from Dr. Qian’s group demonstrated that DGLA can undergo a unique pathway during COX-2-catalyzed peroxidation and produce distinct free radical byproducts. Here we proposed that (1) DGLA’s anti-cancer activity is derived from its distinct byproduct, e.g., 8-hydroxyoctanoic acid (8-HOA), from COX-2-catalyzed peroxidation, and (2) by knocking down cellular D5D expression, we can take advantage of the commonly overexpressed COX-2 in cancer cells to promote 8-HOA formation, inhibit colon cancer growth and migration, and develop a novel cancer therapy and a paradigm shift concept in contrast to classic COX-2 inhibition strategy in cancer treatment. Our results showed that 8-HOA, at physiological concentrations, could suppress human colon cancer cell growth and migration, by serving as a histone deacetylase inhibitor and DNA damage agents. Data also showed that knocking down D5D in colon cancer cells promoted endogenous formation of 8-HOA to a threshold level which then inhibited cancer cell growth and migration. Consistent with the in vitro data, knocking down D5D in human colon cancer cell-derived mice xenograft tumors along with DGLA supplementation promoted endogenous formation of 8-HOA in vivo and significantly suppressed tumor growth. In addition, direct supplementation and endogenous formation of 8-HOA from COX-2 catalyzed DGLA peroxidation were found to enhance the efficacies of various chemotherapeutic drugs. In conclusion, we demonstrated that by taking advantage of commonly overexpressed COX-2 in cancer, D5D knockdown can promote the formation of 8-HOA from DGLA peroxidation to inhibit cancer growth and migration. Results from this work will lead us to develop a novel ω-6 based treatment strategy for colon cancer.
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.
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.
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.
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.
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.

Browse

Browsing Pharmacy Doctoral Work by Title