Short interfering RNAs (siRNAs) trigger RNA interference (RNAi) both in vitro and in vivo, where the expression of the encoded protein is silenced. Its potential use as a therapeutic agent is limited by its rapid enzymatic degradation and low cellular uptake. Therefore, a delivery carrier is desired to increase its solution stability and improve cellular uptake. In this study, Arginine-9 (R9), a cell penetrating peptide derived from the HIV 1 Tat protein, was investigated as a potential carrier for siRNAs at pH 7.3. The optical activity of siRNA decreased with increasing R9 concentration, with only 7.8±3.8% of the initial absorbance at 260nm remained at siRNA saturation. The highest binding ratio of R9 to siRNA determined from the UV/Vis spectra was 10.3:1 (corresponds to a charge ratio of 2.2:1 (+/-)). The measured hydrodynamic diameter increased with increasing R9, with a maximum value of ~1um at siRNA saturation. At R9 to siRNA charge ratios below 5.74:1, the surface charge of the complexes increased rapidly with the addition of R9. However, the rate of increase of Zeta potential decreased significantly with subsequent addition of R9. At charge ratio above 1.43:1, the complexes expressed low surface charge which led to the formation of aggregates in solution. A new peptide library was designed which utilized several properties of known cell penetrating peptides. In vitro siRNA transfection of eGFP siRNA with C166-GFP cells was used to determine the transfection efficiency of the new peptides. Preliminary results of the newly designed peptides showed that some of them are not as effective when compared to Lipofectamine 2000 (Invitrogen). However, this experimental protocol can be extended to study transfection efficiency of the remaining peptides in the library.

Short interfering RNAs (siRNAs) trigger RNA interference (RNAi) both in vitro and in vivo, where the expression of the encoded protein is silenced. Its potential use as a therapeutic agent is limited by its rapid enzymatic degradation and low cellular uptake. Therefore, a delivery carrier is desired to increase its solution stability and improve cellular uptake. In this study, Arginine-9 (R9), a cell penetrating peptide derived from the HIV 1 Tat protein, was investigated as a potential carrier for siRNAs at pH 7.3. The optical activity of siRNA decreased with increasing R9 concentration, with only 7.8±3.8% of the initial absorbance at 260nm remained at siRNA saturation. The highest binding ratio of R9 to siRNA determined from the UV/Vis spectra was 10.3:1 (corresponds to a charge ratio of 2.2:1 (+/-)). The measured hydrodynamic diameter increased with increasing R9, with a maximum value of ~1um at siRNA saturation. At R9 to siRNA charge ratios below 5.74:1, the surface charge of the complexes increased rapidly with the addition of R9. However, the rate of increase of Zeta potential decreased significantly with subsequent addition of R9. At charge ratio above 1.43:1, the complexes expressed low surface charge which led to the formation of aggregates in solution. A new peptide library was designed which utilized several properties of known cell penetrating peptides. In vitro siRNA transfection of eGFP siRNA with C166-GFP cells was used to determine the transfection efficiency of the new peptides. Preliminary results of the newly designed peptides showed that some of them are not as effective when compared to Lipofectamine 2000 (Invitrogen). However, this experimental protocol can be extended to study transfection efficiency of the remaining peptides in the library.

Mitochondria serve as the major source of reactive oxygen species (ROS) production in cells resulting in antioxidant systems and cell signaling pathways that are unique to mitochondria. Thioredoxin-2 (Trx-2) is the mitochondrial member of the thioredoxin superfamily, and acts specifically to reduce the mitochondrial peroxidase, peroxiredoxin-3. It has been proposed that Trx-2 associates with cytochrome c, which functions in mitochondrial respiration and apoptosis. Homozygous Trx-2 deletion in mice is embryonic lethal and it is hypothesized here that Trx-2 lethality is caused by loss of mitochondrial function and oxidative stress. Results of experiments investigating mitochondrial integrity, cell viability, and ROS levels in Trx-2(-/-) mouse embryonic fibroblasts (MEFs), and results from Trx-2 siRNA MEFs, are similar to findings of knockouts in previously reported proteins that function in mitochondrial respiration and support the involvement of Trx-2 in this process. Mitochondrial ROS have also been implicated as major secondary messengers in cell signaling. Results reported here using cancer cells and cancer cells depleted of mitochondrial DNA, which consequently produce few ROS, have indicated that mitochondrial ROS produced in hypoxia are necessary for HRE and ARE activation, and are fundamental in the activation of SP-1 during reoxygenation. However, mitochondrial ROS are not required for HIF-1α protein expression in hypoxia, indicating a unique relationship between HIF-1α, hypoxia, and mitochondrial ROS.

No description available.

In many tumors, persistently-active signal transducer and activator of transcription 3 (STAT3) imparts several oncogenic features such as survival, proliferation, angiogenesis, and immune escape. Therefore, STAT3 targeting in cancer and cancer-exposed dendritic cells (DCs) is important for cancer therapy. Our objective is developing delivery modalities of STAT3-targeting small interfering RNA (siRNA) using lipid-modified polyethylenimine (PEI) polyplexes and poly(D,L lactic-co-glycolic) acid (PLGA) nanoparticles (NPs), and evaluating the therapeutic outcomes in vitro and in vivo. Significant increase in siRNA condensation, protection, and cellular uptake by B16.F10 melanoma was seen by stearic-acid-modified PEI (PEI-StA) compared to unmodified PEI. Moreover, PEI-StA increased the STAT3 silencing potency of siRNA compared to PEI. STAT3 knockdown was accompanied with significant induction of interleukin-6 (IL-6) secretion and reduction of vascular endothelial growth factor (VEGF) production and cytotoxicity evidenced by increased Caspase 3 activity in vitro and in vivo, and significant inhibition in tumor growth. Analysis of tumor microenvironment showed CD3+ cells infiltration corresponding to STAT3 knockdown. The levels of CD4+ helper cells, CD8+ cytotoxic cells, and NKT cells significantly increased. DC infiltration and activation significantly increased in tumor mass following STAT3 knockdown as evidenced by high expression of CD86 and CD40. Moreover, IFN-, IL-12, and TNF- significantly increased following STAT3 knockdown by PEI-StA compared to PEI, suggesting Th1-type immunity. Allogenic capacity of DCs isolated from siRNA-treated mice was evidenced by the high T cell proliferation and IL-2 production in mixed lymphocytes reaction (MLR). Then, we explored STAT3 knockdown in DCs exposed to tumor derived factors (TDFs). We investigated encapsulation of siRNA complexes (PEI or PEI-StA) into PLGA NPs (PLGA-P and PLGA-PS). PLGA-P and PLGA-PS had an average diameter of ~ 370 nm and zeta potential of ~ -16 mV. Uptake and endosomal localization was confirmed. After TDFs exposure, DCs showed high STAT3 and low CD86 expression. STAT3 silencing by PLGA-P and PLGA-PS restored DC functionality as evidenced by upregulation of CD86, IL-12, and TNF- and MLR activity. PLGA significantly reduced PEI-associated toxicity. Therefore, STAT3 targeting in B16 cells by siRNA polyplexes of PEI and PEI-StA, or in DCs by PLGA-P and PLGA-PS provide potential strategies for cancer therapy. / Pharmaceutical Sciences

The ability of small interfering RNA (siRNA) to silence specific target genes offers not only a tool to study gene function but also represents a novel approach for the treatment of various human diseases, including cancers. The clinical use of siRNA, however, has been severely hampered by the inefficient delivery of these molecules to target cell populations due to their instability, inefficient cell entry, and poor pharmacokinetic profile. Much effort has therefore been devoted to the development of efficient in vivo siRNA delivery systems, with liposomes being the most widely employed vector. The traditional methods of packaging siRNA into liposomes, however, are often quite complex and labour-intensive, with the resulting products also being unstable at room temperature which limits their wide spread application in the clinic. The main aim of this research was to develop a simple, yet efficient, formulation technique to prepare stable siRNA-loaded liposomes which could be utilized as an efficient therapy for cancer treatment. Throughout this study, cervical cancer was used as the model system to assess the efficiency of various delivery systems. It is an ideal disease for siRNA therapy due to the cancer’s reliance on the expression of a single messenger RNA sequence which encodes two essential viral oncogenes, E6 and E7. Previous research has shown that targeting E6 and E7 by siRNA in cervical cancer cells in vitro results in either cell senescence or apoptosis. This thesis investigates the feasibility of applying E6/7 siRNA both intravaginally and intravenously to model the treatment of early-stage and end-stage cervical cancer, respectively. The practicability of applying E6/7 siRNA intravaginally for the treatment of localised cervical cancer tumours was firstly evaluated by administrating liposome-complexed siRNAs directly into the vaginal cavity of transgenic E7 mice. As no knockdown of E7 in cervical epithelium was observed for mice which received repeated treatments of E6/7 siRNA, the vaginal delivery efficiency of liposomes was further examined using fluorescently-labelled oligonucleotides. Contrary to previous reports, no delivery of lipoplexes into cervicovaginal tissues was detected irrespective of the dosage, type of lipid vector used, or the mouse estrus state at the time of administration. This lack of delivery was likely due to the poor retention of lipoplexes in the vaginal cavity as well as the inefficient penetration of lipoplexes across the mucosal layer lining the cervicovaginal epithelium. Overall, these findings indicated the necessity of developing more suitable and clinically acceptable vaginal siRNA delivery systems to enable this treatment strategy to become a reality. Despite the challenges of using liposomes to deliver siRNA via vaginal administration, their successful use in delivering siRNA intravenously to tumours was demonstrated in a subcutaneous cervical cancer mouse model. These experiments were carried out using PEGylated siRNA-loaded liposomes which were formulated using a novel Hydration-of-Freeze-Dried-Matrix (HFDM) technique. Compared to the existing formulation strategies, this method of preparation is less labour-intensive and the end product is also freeze-dried, ensuring product stability. It was found that the liposomes prepared using the HFDM method were stable in the presence of serum and they also possessed high siRNA entrapment and gene-silencing efficiencies. Following intravenous administration to mice, these particles were also found to accumulate in subcutaneous tumours to a similar degree compared to formulations prepared using a previously established technique. Importantly, these HFDM-formulated preparations showed superior stability over ones prepared using the traditional formulation method, with the particles still retaining 100% of their gene-silencing ability after storage for one month at room temperature. Using HFDM-formulated liposomes loaded with siRNA against Green Fluorescence Protein (GFP), a 50% knockdown of the GFP expression was achieved in tumours following intravenous administration. Additionally, the use of E6/7-targeted siRNA also resulted in a 50% reduction in tumour size when the siRNAs were delivered using HFDM-formulated liposomes. Importantly, this level of tumour growth suppression was comparable to that achieved from cisplatin, a clinically used chemotherapeutic for cervical cancer, at the clinically used dose. Overall, this research demonstrated that while there are still some challenges to overcome for siRNA to be used vaginally for cervical cancer treatment, HFDM-formulated PEGylated liposomes showed promise in bringing E6/7 siRNA forward as a treatment option for end-stage cervical cancer. In addition, the simplicity of preparation procedure along with superior product stability obtained from the HFDM method developed in this thesis will likely facilitate the translation of siRNA technology from laboratory to clinics for a range of other medical applications.

Hepatic fibrosis is a serious health problem in many parts of the world. However, its treatment remains severely limited because of inadequate target specificity. HSC are the largest reservoir of vitamin A in the body. They are also the principal players responsible for the pathogenesis of liver fibrosis. Targeting HSC is an effective strategy for treatment of liver fibrosis. The specific association of BMP4 with various liver diseases including liver fibrosis makes it an ideal candidate for targeting HSC cells using siRNA. The objective of this study is to develop and characterize vitamin A (VA)-coupled liposomes for the targeted delivery of BMP4-siRNA to cultured HSC. DOTAP/DOPE liposomes surfaces were prepared by thin film hydration and their surfaces were decorated with VA (1:2 mol/mol). Particle size and zeta potential were determined using ZetaPALS. In addition, the siRNA binding efficiency was determined by ultra-centrifugation and fluorescence assays. The cytotoxicity of VA-conjugated liposomes was evaluated by the WST-1 cytotoxicity assay. Inhibition of BMP4 and α-SMA was determined by real time PCR and ELISA. Their average particle size was in the range of 100-120 nm and they exhibited zeta potential around +45 mV. VA-coated liposomes were mixed with BMP4-siRNA, forming lipoplexes with particle sizes less than 200 nm and zeta potential around +25 mV. The presence of VA did not alter the siRNA binding efficiency, it also had no effect on cytotoxicity, but resulted in enhanced cellular uptake of siRNA as shown by flow cytometry. There was a significant reduction in BMP4 mRNA with VA-coupled liposomes carrying BMP4-siRNA. Moreover, BMP4 gene silencing was accompanied by a significantly reduced the expression of the potent fibrinogenic α-SMA at mRNA and protein levels. In conclusion, VA-coated liposomes were successfully able to target and deliver BMP4-siRNA to HSC. This could offer an interesting perspective for the treatment of liver fibrosis. / February 2016

The aryl hydrocarbon receptor (Ah-receptor) is a member of the bHLH-PAS protein family. The Ah-receptor is a ligand dependent transcription factor, which activates a wide range of genes, most notably the xenobiotica metabolising genes, CYP1A1 and CYP1A2. The biological function of the Ah-receptor is still unknown and an endogenous ligand has yet not been identified. A possible Ah-receptor ligand is 6-formylindolo[3,2-b]carbazole (FICZ). FICZ has a high affinity for the Ah-receptor and is rapidly metabolised by CYP1A1, CYP1A2 and aldehydeoxidase (AOX). To try to trap FICZ or other possible endogenous Ah-receptor ligands, the metabolising enzymes CYP1A1, CYP1A2 and AOX were blocked. This was achieved through chemical blockage of CYP1A1 and CYP1A2 by ellepticin and through silencing with siRNA directed against CYP1A1 and CYP1A2. Successful blockage would be seen as an increase in Ah-receptor dependent XRE-luciferase activity. Chemical blockage of AOX with tungstate did not affect FICZ-dependent XRE-luciferase activation which could indicate that HepG2 cells lack AOX. The chemical blockage of CYP1A1 and CYP1A2 with ellepticin modified the XRE-luciferase response, but did not completely block Ah-receptor activation. In addition it is possible that ellepticin is a ligand for the Ah-receptor. The blockage of CYP1A1 by siRNA was successful; a silencing of CYP1A1 mRNA by at least 50 percent was detected. However due to lack of time it was not tested if the blockage of CYP1A1 and CYP1A2 was sufficient to trap Ah-receptor ligands.

RNA interference represents a useful tool for modulating expression of sarcosine metabolism genes and for studying the role of sarcosine in prostate cancer. The thesis "Modulation of sarcosine metabolism by RNA interference" summarizes current state-of-the-art of possible regulation of gene expression using various types of nucleic acids. Furthermore the thesis also deals with the issue of the transfer of these regulatory agents to target tissues and finally it describes sarcosine including its involvement in the metabolic cycles of the cell. The main aim of the experimental part of this work was to determine the influence of knock down sarcosine dehydrogenase (SARDH) on other enzymes involved in sarcosine metabolism. This effect was assessed by determining the gene expression of individual genes encoding the four major sarcosine pathway enzymes by quantitative real-time PCR analysis. Experiments were performed on three different prostatic cell line types, PNT1A, DU-145 and PC3. The most significant differences on level of gene expression were observed in carcinoma cells DU-145 in which a significant increase in gene expression of dimethylglycine dehydrogenase (DMGHD) and sarcosine oxidase (PIPOX) was observed after applications of siRNA targeting the SARDH.

No description available.