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Evaluation of Reconstituted High Density Lipoprotein as an Anticancer Drug Delivery Platform
No full textTargeted Nanoparticles for the Treatment of Neuroblastoma
No full textNeuroblastoma (NB) is one of the most frequently diagnosed tumors in infants and children. However, the mechanism by which it is initiated and subsequently develops on the molecular and cellular level is yet to be fully elucidated. Its wide spectrum of clinical presentation has baffled physicians and biomedical scientists alike. The variant called high risk neuroblastoma (HRNB) is extremely resistant to the currently available drug regimes. Despite the recent advances in anti-cancer agents and the use of multi-modality therapy for the treatment of HRNB the morbidity and mortality in this group of patients continues to remain high.
The purpose of our project was to find novel alternative therapeutic approaches by encapsulating known anti-NB agents in a lipoprotein based formulation to achieve selected, targeted delivery of these drugs to HRNB tumors. We wanted to enhance the therapeutic efficacy of these drugs that have shown encouraging results in pre-clinical trials but have so far exhibited an adverse pharmacokinetic profile precluding their systemic application. Our laboratory has been working for the last several years on a novel drug delivery platform by encapsulating drugs into the core of high density lipoprotein (HDL) type nano-particles. Using this strategy, we encapsulated all-trans retinoic acid (ATRA), fenretinide (FR) and valrubicin into reconstituted HDL (rHDL) nanoparticles and subsequently evaluated some of their physical and chemical properties and their anti-NB potential. Further, we tested the efficiency of an apolipoprotein mimetic peptide called 5-A peptide as a component of rHDL particles and compared its efficiency with apolipoprotein A-1 (Apo-A1). The 5-A peptide offers numerous advantages over the Apo-A-1 both in terms of cost of production as well as manufacturing time.
After successfully encapsulating the drugs, we characterized them and tested their cytotoxic potential on various cancerous cell lines. We also conducted cell uptake studies to test our hypothesis of tissue targeting and selective uptake of rHDL nano-particles mediated by the scavenger receptor type B1 (SR-B1). We conducted a pilot study on nude mice in which we administered rHDL containing fluorescent dye intravenously in mice xenografted with NB tumors and took subsequent images to track its distribution in the body. Our results demonstrate that it is possible to encapsulate ATRA, FR and valrubicin into rHDL preparations with a predictable efficiency; these nano-particles show a dose dependent cytotoxic effect on NB cell lines. We anticipate that the results of our studies will facilitate the application of liposomal nano-particles and these novel drugs in the treatment of HRNB in the future
Optimization of Reconstituted High Density Lipoprotein nanoparticles as a Delivery System for Neuroblastoma
No full textDespite many advances in cancer therapy over the last few decades, cancer remains one of the most common causes of death in not only the United States, but around the world. Two of the major problems cancer patients face today are the horrific side effects associated with chemotherapy, and the development of drug resistance. Both of these become even bigger problems when they are applied to children. Neuroblastoma is one of the most common forms of pediatric cancer. High risk Neuroblastoma patients are commonly faced with intensive multi-modal therapies in attempt to overcome a very aggressive disease. Due to the intensive therapy required, side effects can often linger even after remission is achieved in these patients, and multi-drug resistance is common due to the high levels of Doxorubicin administered. New solutions are needed in order to overcome both of these problems in Neuroblastoma as well as other types of cancer.
In this thesis, we studied the effects of different formulation and preparation techniques for the reconstituted high density lipoprotein nanoparticle model for anti-cancer agent delivery. During these studies we found that naturally derived mixes of phosphatidylcholine, and lower levels of apolipoprotein A-1 increase the encapsulation efficiency of the rHDL nanoparticles. We also determined that the addition of lyophilization during preparation before cholate dialysis, forms a more homogeneous preparation. After the optimization of the particle formulation and preparation, we tested the efficacy of two model anti-cancer agents in different cancer cells. First we showed the ability of the rHDL-siRNA nanoparticles to knockdown the SR-B1 protein is greater than the knockdown of a commercial transfection kit. Finally we prove that the rHDL also improves the cytotoxic efficacy of a novel treatment for Neuroblastoma involving Imatinib Mesylate and Saquinavir.
In conclusion, the results of this thesis show a more detailed knowledge of the rHDL nanoparticle formulation as well as how it can be applied as an effective delivery system for both siRNA and chemotherapeutic agents. This data should help push our formulations closer to clinical applications, and toward helping reduce the toxic side effects of many chemotherapeutic agents, as well as reducing the incidence of drug resistance
Immunoreactivity of Lecithin: Cholesterol Acyltransferase (LCAT); A Tool for Measurement of Levels and Characterization
Full text linkMurray, Karen R., Immunoreactivity of Lecithin:Cholesterol Acyltransferase (LCAT); a Tool for Measurement of Levels and Characterization. Doctor of Philosophy (Biomedical Sciences), August, 2000, 162pp., 12 tables, 30 illustrations, bibliography, 150 titles. Lecithin:cholesterol acyltransferase is secreted by the liver into the plasma where it catalyzes the esterification of high density lipoprotein (HDL) cholesterol as part of the reverse cholesterol transport pathway. Via this pathway both HDL and LCAT have been linked to reducing the risk of atherosclerosis and coronary heart disease. These studies seek to develop an immunoassay to measure LCAT mass and to use immunoreactivity to elucidate the contribution of the highly conserved 121-136 domain of LCAT toward enzyme structure/function and HDL interaction. Several immunoassay models and antibody combinations were investigated to develop an ELISA assay for LCAT. Solid phase immunoassays were found to be most suitable for measuring LCAT from cell culture medium and in partially purified preparations. The immunoassay was analyzed for matrix interference, recovery studies, intra-run precision and inter-run precision. Evaluation of the immunoassay models and antibodies was extended to determine the potential for application toward measuring LCAT in plasma; however, the antibodies screened lacked the needed sensitivity. Studies were conducted to characterize the 121-136 region, a putative lipoprotein substrate binding domain. Differential immunoreactivity was demonstrated for a site directed antibody against the 121-136 region, in contrast to antibodies directed against the entire LCAT molecule, when the enzyme was bound to a hydrophobic surface or to substrate HDL, but not when bound to an alternate antibody. Three naturally occurring mutants within the 121-136 region, were tested for immunoreactivity with the same panel of antibodies and compared to wild type enzyme. These studies demonstrate that the 121-136 region of LCAT resides on the surface of the enzyme that has a high affinity for hydrophobic surfaces and mutation within this region significantly affects the exposure of different epitopes. This suggests that this region could plan an important role in enzyme interaction with its hydrophobic lipoprotein substrates and that mutations within this region could alter enzyme conformation affecting substrate interaction
Quantitative Proteomic Investigation of Estrogenic Endocrine-Disrupting Effects in the Rat Uterus
No full textThe mammalian uterus is one of the most sensitive organs for estrogenicity. However, the widely used rat uterotrophic assay to assess known and potential estrogenic compounds only considers the uterine wet weight gain as an endpoint measurement. To complement this method with an advanced technology that reveals molecular targets, we analyzed changes in protein expression using label-free quantitative proteomic analysis by liquid chromatography–mass spectrometry from uterine protein extracts of ovariectomized rats after daily 17β-estradiol exposure for five days. We performed shotgun proteomic analysis of the uterus to identify candidate proteins for use as markers of estrogenicity. In addition, we mapped the differentially expressed proteins from untargeted analysis to signaling networks and biological processes through Ingenuity Pathway Analysis. We selected twelve of the top up- and down-regulated proteins for further evaluation by selected reaction monitoring-based targeted quantitation. Of the final six candidate markers, we verified all six as markers of estrogenicity by the application of the panel to testing rats exposed to a low and high dose of the known estrogenic compound bisphenol A. Altogether, the results of this study demonstrate the power of combining untargeted and targeted quantitative proteomic methods for a comprehensive analysis in rat uterus to evaluate changes in protein expression levels due to estrogen exposure, and to uncover candidate markers of estrogenicity in the development of a targeted proteomics panel
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The Teratogenic Effects of Nocodazole and Acrylamide in Mus Musculus
Full text linkIn two separate experiments, weight adjusted doses of nocodazole and acrylamide were injected intraperitoneally at various time intervals into twelve week old female mice. Within the nocodazole experiment, the doses were injected at varying time intervals before and after mating. On day seventeen of gestation, the female mice were sacrificed and their uterine contents examined. Nocodazole induced a significant increase in reproductive pathology per total implants when administered one hour after mating to the (SECxC57BL)F, stock: 5.00% total deads, 70.23% moles, and 3.41% abnormal fetuses. Acrylamide treatment produced a significant reduction in live births when administered six hours after mating: 50.86% moles and 46.46% living fetuses per total implants
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Studies on Poly (ADP-ribose) Synthesis in Lymphocytes of Systemic Lupus Erythematosus Patients
Full text linkA method for assaying poly (ADP-ribose) polymerase (PADPRP) activity in lymphocytes of systemic lupus erythematosus (SLE) patients has been developed. Using this method, PADPRP activity has been studied in lymphocytes from 15 patients and 13 controls. The mean activity in SLE lymphocytes was significantly lower than that in controls and 60% of the SLE patients demonstrated activities below the minimum of the control population. Possible mechanisms for this altered metabolism were investigated. The Km app of PADPRP for NAD; size distribution, branch frequency, and rates of turnover of polymers; competition for substrate; and number of PADPRP molecules were studied. The data demonstrated that SLE lymphocytes have a decreased synthetic capacity rather than alterations in the substrate or in turnover of the product
Enhancing the Solubility of Valrubicin via Albumin and TPGS Formulations
Full text linkHuman serum albumin (HSA) and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) are versatile biocompatible materials used in drug formulation. Due to its lipophilicity, the anticancer drug valrubicin (VALSTAR) has been solubilized with Cremophor EL, a solvent known for its systemic toxicity. While valrubicin is less toxic than its widely used hydrophilic anthracycline analogues, its clinical use is currently restricted to intravesical route for bladder cancer treatment. Because preliminary studies have shown a strong affinity of valrubicin for HSA and TPGS micelles, this study was aimed to explore the potential of reduced HSA (rHSA) or TPGS as excipients for valrubicin. In an aqueous environment, valrubicin solubility was enhanced from 0.1 to 85.4% using rHSA while it was dependent upon TPGS concentration. With appropriate formulation approaches, rHSA or TPGS could serve as valrubicin transporters and could thus, enable its systemic administration and extended use beyond bladder cancer to other cancer types
Characterization and Optimization of Nanoparticles for Polynucleotide delivery
No full textNucleic acid therapeutics involves the use of polynucleotides (DNA, RNA) as novel therapeutic agents for the treatment of a wide range of diseases including cancer and several metabolic and genetic disorders. However, the highly unstable nature of RNA molecules necessitates the use of drug carriers to prevent them from nuclease degradation and facilitate targeted delivery in vivo. Hence, this study was conducted to optimize the preparation of nanoparticle carriers in order to improve the stability of the polynucleotides (siRNA and mRNA). Additionally, as heterogeneity and stability of nanoparticle formulations are major issues preventing the clinical approval of therapeutic formulations this study was also focused on improving the homogeneity and the stability of the nanoparticles. In the siRNA study, reconstituted high density lipoprotein (rHDL) nanoparticles were used as the delivery vector. Optimization of siRNA-rHDL formulation was attempted with respect to homogeneity, size of the nanoparticle and entrapment efficiency of siRNA. The results showed that the inclusion of the lyophilization step in the preparation of nanoparticles resulted in a marginal increase in the homogeneity. The size analysis of siRNA rHDL nanoparticles using AFM and TEM imaging revealed the presence of spherical nanoparticles in the range of 10-16nm. Optimization studies with mRNA peptide nanoparticle formulation were conducted using a combination of cationic detergents and peptides at varied concentrations. The particle size analysis via Dynamic Light Scattering (DLS) detector revealed the presence of 268 nm diameter particles as the major component of the mRNA nanoparticle formulation that involved the combination of DOTAP (neutralizer) and Myr-5A (Apo A-I mimetic peptide). Further optimization of this formulation will be required to improve the homogeneity of the nanoparticles
Identification of Oxidized Proteins in Alzheimer's Disease
No full textJoungil Choi, Identification of Oxidized Proteins in Alzheimer’s Disease. Doctor of Philosophy (Molecular Biology and Immunology). August, 2002. Pages-110. Tables 8. Figures 24. Oxidative modification of specific proteins is central to the pathology of Alzheimer’s disease (AD). The purpose of this study was to identify the oxidation-sensitive proteins in neuronal cells, fibroblasts from AD subjects, and in the blood of AD patients. In all cases, age-matched non-Alzheimer’s samples were used as controls. Proteomic methods were used to isolate and characterize the oxidized proteins. These included two-dimensional gel electrophoresis, immunolocalization of oxidized proteins and identification by MALDI-TOF mass spectroscopic methods. It was hypothesized that knowledge of these critical oxidation-sensitive proteins would shed light on the underlying mechanism of the disease. In addition, it was postulated that these proteins might prove to be biomarkers for early detection and monitoring the progress of the disease. The results show that two different oxidative stressors (H2O2 generated enzymatically, or the amyloid beta peptide, AB25-35) induce apoptotic cell death and oxidation of specific proteins (heat shock protein 60 and vimentin) in skin fibroblasts from AD subjects and in neuronal cells. In addition, the results indicate that susceptibility of these two proteins to oxidative stress is increased in fibroblasts from AD patients, compared to non-AD controls. Pretreatment with antioxidants (e.g., vitamin E or flavonoids) protect these proteins from oxidative damage. Both heat shock protein 60 and vimentin, have been suggested to function as antiapoptotic proteins. Thus, their oxidative damage could lead to the apoptotic neuronal cell death in Alzheimer’s disease. In the blood plasma of AD subjects, isoforms of fibrinogen gamma chain and alpha-1 antitrypsin were found to be oxidized. These proteins exhibited to a two- to six-fold greater specific oxidation index in plasma from AD subjects when compared to controls. Both of these proteins have been suggested to be implicated in oxidation-mediated damage of inflammation in the AD brain
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