Browsing by Subject "siRNA"
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Item Characterization and Optimization of Nanoparticles for Polynucleotide delivery(2015-05-01) Conjeevaram Nagarajan, Bhavani Saranya; Lacko, Andras G.; Cistola, David P.; Krishnamoorthy, Raghu R.Nucleic 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.Item North Texas Health & Science - 2011, Issue 2(University of North Texas Health Science Center at Fort Worth, 2011-01-01)Item Optimization of Reconstituted High Density Lipoprotein nanoparticles as a Delivery System for Neuroblastoma(2013-12-01) Hinze, Cheryl L.; Lacko, Andras G.Despite 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.Item Role of Glucocorticoid Receptor β in Glucocorticoid-Induced Ocular Hypertension(2008-01-01) Jiang, Ming; Yorio, Thomas; Krishnamoorthy, Raghu R.; Dibas, AdnanJiang, Ming, Role of Glucocorticoid Receptor β in Glucocorticoid-Induced Ocular Hypertension, Master of Science. (Biomedical Sciences). Purpose: Previous studies from our laboratory have shown that glaucomatous trabecular meshwork ™ cells have a lower expression of glucocorticoid beta (GRβ) compared to normal TM cells. Overexpression of glucocorticoid receptor β was found to attenuate glucocorticoid responsiveness in glaucomatous TM cells. The purpose of this study was to determine if downregulating GRβ expression could increase glucocorticoid responsiveness in cultured transformed normal trabecular meshwork cells (NTM5 cells) and primary trabecular meshwork cell lines derived from normal individuals. Methods: siRNA oligonucleotide sequences specific for GRβ were designed, cloned into expression vectors and sequenced. TM cells were transfected with different siRNA constructs for GRβ, while a scrambled sequence served as a negative control. Immunoblot analyses were carried out in the transfected TM cells to determine knockdown of GRβ expression. In another set of experiments TM cells were transfected with either a GRβ siRNA construct or an empty vector (control) and co-transfected with a GRE-luciferase reporter and a SV40 promoter-β galactosidase construct to normalize for efficiency of transfection. Promoter-reporter assays were carried out to determine the effect of GRβ knockdown on glucocorticoid-mediated luciferase reporter activity. Results: Using MetafecteneTM rpo as the transfection reagent for siRNA, an appreciable 50% to 90% decrease in GRβ protein expression was observed 72 hours post-transfection in three GRβ siRNA constructs transfected in NTM5 cells, compared to the scrambled sequence transfected cells. NTM5 cells transfected with the empty vector (control) showed a 3.5 fold increase in luciferase activity after dexamethasone treatment, which was further increased (4 to 5 fold) by knocking down GRβ expression using a siRNA construct specific for GRβ. A similar trend was found using GRβ-siRNA#3 to transient knockdown GRβ in four primary TM cell lines – NTM210-05, NTM486-04, NTM174-04, and NTM153-00, respectively. To investigate to glucocorticoid responsiveness in cells permanently transfected for knockdown of GRβ, we made 6 clones from NTM5 cells. There were 10 to 20 fold induction in luciferase activity in GRβ siRNA stably transfected NTM5 cells, following glucocorticoid administration, respectively. Conclusion: In this study, using RNAi(s) which is specific for GRβ, we decreased GRβ expression in vitro in several different cell lines. Using luciferase assays it was found that the glucocorticoid responsiveness after knockdown of GRβ in vitro in trabecular meshwork cells was significantly enhanced. Decreased GRβ expression significantly increased glucocorticoid responsiveness not only in normal transformed human trabecular meshwork cells (NTM5 cells), but also in four primary trabecular meshwork ™ cell lines derived from normal individuals. These results further support the notion that GRβ negatively regulates responsiveness in TM cells. Keywords: glaucoma, glucocorticoid, glucocorticoid receptor beta, trabecular meshwork