Pharmacy

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

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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.
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.
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 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.
Dual-Functionalized Liposomes for Gene Delivery to Brain to Prevent and Treat Alzheimer’s Disease
(North Dakota State University, 2019) dos Santos Rodrigues, Bruna
Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder which lacks effective disease-modifying therapies. We have investigated the therapeutic potential of pDNA encoding apolipoprotein E2 (ApoE2), or nerve growth factor (NGF) by transporting pDNA across the blood brain barrier (BBB) and expressing the ApoE2 or NGF into brain, using brain-targeted liposomal nanoparticles for treatment of AD. We explored the neuroprotective functions of ApoE2 and survival-promoting properties of NGF through gene therapy as potential disease-modifying therapies for AD. We designed brain-targeted gene delivery systems with prolonged systemic circulation and enhanced cellular penetration by conjugating transferrin (Tf) ligand and cell-penetrating peptide (CPP) to liposome via DSPE-PEG phospholipid. In vitro characterization studies showed that the nanoparticles had homogeneous particle size, positive zeta potential and protected plasmid DNA against enzymatic degradation. Additionally, they exhibited low hemolytic potential and low cytotoxicity. Cellular uptake occurred in a time-dependent manner through multiple endocytosis pathways. Reporter gene transfection and consequent protein expression in different cell lines were significantly higher using CPP-Tf-liposomes compared to single modified liposomes. The ability of these liposomes to escape from endosomes can be an important factor which may have likely contributed to the high transfection efficiency observed. In vivo brain targeting efficiency of designed liposomes was evaluated using in vitro triple co-culture BBB model. Dual-modified liposomes efficiently crossed in vitro BBB and, subsequently, transfected primary neuronal cells. Increasing NGF expression in primary neuronal cells following treatment with liposomes increased the levels of pre-synaptic marker synaptophysin in vitro. PenTf-liposomes containing pDNA efficiently induced protein expression in the brain of mice. A dose response study was performed in order to select the appropriate dose of pNGF to induce significant NGF expression and, consequently, a therapeutic effect. Administration of PenTf-liposomes containing pNGF to APP/PS1 mice (aged 3 months) for four weeks (one injection per week) decreased the levels of toxic soluble and insoluble Aβ peptides. Additionally, the treatment stimulated cell proliferation and increased the levels of synaptic markers, synaptophysin and PSD-95. These data suggest the therapeutic potential of PenTf-liposome-mediated NGF gene therapy which can be considered as a candidate for treatment of AD.
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.
Elicit Anti-Cancer Effects of Dihomo-γ-linolenic Acid by Exploiting Overexpressed COX-2 in Cancer: Development of an ω-6 Fatty Acid-Based Therapeutic Strategy for Pancreatic Cancer
(North Dakota State University, 2017) Yang, Xiaoyu
Video summarizing Ph.D. dissertation for a non-specialist audience.
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.
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.

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