No description available.

No description available.

<p>Electric field mediated gene delivery (EFMGD) or electrotransfection is a popular, non-viral gene delivery method that has been used in a variety of studies and applications ranging from basic cell biology research to clinical gene therapy. Yet, the mechanism(s) by which electrotransfection facilitates DNA delivery across the cell membrane into the cell and its subsequent intracellular transport across the cytosolic space towards the nucleus have been insufficiently studied and still remain controversial. Understanding these mechanisms and characterizing the intracellular journey of pDNA is important for understanding the physiological barriers of EFMGD within the cell, which can be used to engineer better solutions to overcome these barriers with the ultimate goal of improving the transfection efficiency of this technology. </p><p>Conventional thought in the field assumes that such transport modes as diffusion, electrophoresis, and electro-osmosis, which govern the entry of small molecules into cells through electric field-generated transient membrane pores, also apply to electric field-mediated delivery of therapeutic DNA. We propose that electrically-induced gene transfer into cells is governed by an alternative, more active mode of transport that entails the involvement of cellular endocytic processes. It is our hypothesis that pulsed electric field generate these membrane pores which interact with nearby DNA molecules; but that actual DNA translocation across the membrane is driven by endocytosis, which consequently, then, also plays a role in the intracellular transport of the DNA. To this end, we first investigated the dependence of electrotransfection efficiency (eTE) on binding of plasmid DNA (pDNA) to plasma membrane. Binding concentrates DNA molecules in the vicinity of the cell membrane, which should theoretically result in a greater number of DNA-membrane interactions during pulsed electric field, more internalized DNA, and ultimately, higher eTE values. We demonstrated that supplementing the electrotransfection buffer with divalent cations (Ca2+ and Mg2+) is an effective method of promoting pDNA adsorption to the cell membrane. This cation-mediated increase in DNA adsorption to the cellular membrane resulted in a consequent increase in eTE, up to a certain threshold concentration for each cation. To determine the timeframe for completion of pDNA internalization following pulse treatment, trypsin treatment was applied to cells at different timepoints after electrotransfection to strip off any residual, membrane-bound pDNA that had not been internalized. Trypsin treatment at 10 min post electrotransfection still resulted in a significant reduction in eTE, indicating that the time period for complete cellular uptake far exceeded the lifetime (~ 10 msec) of electric field-induced transient pores. The role of endocytosis was further probed by noting the effect on eTE when cells were treated with three endocytic inhibitors (chlorpromazine, genistein, dynasore) targeting different internalization mechanisms or silenced of dynamin expression using specific, small interfering RNA (siRNA). siRNA silencing and all three pharmacological inhibitors yielded substantial and statistically significant reductions in the eTE. Taken together, these findings suggest that the mechanism of electric-field mediated DNA internalization entails: (i) binding of pDNA to cell membrane and (ii) endocytosis of membrane-bound pDNA.</p><p>The same strategies of pharmacological endocytic inhibition and siRNA silencing was used to further explore and compare electric field-induced pDNA internalization in additional cell lines that differ in terms of cell type, proliferation rates, proliferative capacity (i.e. primary versus immortalized/cancer line), etc. in order to determine whether endocytosis is a universally implicated mechanism across many cell lines. Results showed different endocytic pathways to be recruited for pDNA uptake in a cell-dependent manner and that one or multiple pathways may contribute to uptake within a cell line. </p><p>Taken together, the studies presented in this dissertation provide both indirect and direct evidence suggesting an endocytic role in the translocation of pDNA across the cell membrane and its intracellular routing towards the nucleus for EFMGD. These seminal findings could potentially lead to better understanding of the intracellular barriers encountered by EFMGD, more strategic optimization of electrotransfection parameters than the trial-and-error approach currently used, and enhanced transfection efficiencies.</p> / Dissertation

Damage to peripheral nerves following trauma or disease has a number of consequences including burning pain, muscle wasting, paralysis, or organ dysfunction. The most common form of neuropathy is that associated with metabolic abnormality, notably diabetes. Many diabetics, especially those with poor blood sugar control, ultimately develop a distal symmetrical and painful neuropathy that initially affects the longest peripheral axons, but with time spreads proximally. Deficiency in neurotrophic support has been proposed to contribute to the development of diabetic neuropathy. Recently, peripheral gene delivery of vascular endothelial growth factor (VEGF), neurotrophin-3 (NT-3), NGF, BDNF or hepatocyte growth factor (HGF) has been shown to facilitate the continuous production of neurotrophic factors and alleviate the diabetic neuropathy. The role of glial cell-derived neurotrophic factor (GDNF) in the pathogenesis and therapeutics of diabetic neuropathy is not well defined. The main objectives of this research sought to inspect the protective effect of GDNF peripheral gene delivery during hyperglycemia- or constriction- induced sciatic nerve injury in rats. In present proposal, we propose to investigate the change in organization and expressions of GDNF signaling complex in the sciatic nerve following injury in the initial stage. Subsequently, the recombinant adenovirus was used gene delivery system for GDNF to evaluate the potential of intramuscular administration of gene delivery for prevent nerve degeneration, and the molecular mechanism of GDNF to ameliorate neuropathy will be clarified. The above study would enable us to test the hypothesis that the topical gene delivery might be a suitable strategy for the treatment of diabetic neuropathy and other disorders in peripheral nerve. Furthermore, the results of animal studies might be extrapolated for future clinical application.

Hepatocellular carcinoma (HCC) is one of the most common cancerous diseases worldwide. The annual occurrences exceed one million peoples affected. Currently, the treatment modalities for HCC include surgical resection, trans-arterial embolization (TAE) and chemotherapy. However, these modalities are not completely effective, underscoring the need for development of novel therapeutic approaches. PTEN, a tumor suppressor that antagonizes the PI3K pathway, is frequently mutated or deleted in various human cancers. Loss of PTEN occurs in 40-50% of surgical resected HCC samples and predicts poor prognosis for HCC patients, suggesting PTEN restoration may constitute a treatment alternative for HCC. Since PTEN increased ethanol-induced cytotoxicity in hepatoma cells, PTEN gene delivery may serve as an adjuvant therapy in conjunction with ethanol TAE for HCC. In the present study, we evaluated the efficacy of PTEN gene therapy and its combination with ethanol in a syngenic Novikoff hepatoma model by implantation of N1-S1 cells into livers of Sprague Dawley rats. Adenovirus encoding PTEN (Ad-PTEN) or green fluorescent protein (Ad-GFP) was generated for gene delivery studies. The optimal condition for adenovirus vectors to infect N1-S1 cells was determined at multiplicity of infection (MOI) of 100-200. Infection of N1-S1 cells with Ad-PTEN, but not Ad-GFP, increased PTEN levels and led to 40-50% inhibition of cell proliferation via cell cycle arrest. Besides, the half maximal -inhibitory concentration (IC50) for ethanol in N1-S1 cells was determined at 6%. Combination with PTEN gene delivery further augmented the cytotoxicity of ethanol in N1-S1 cells from 40% to 70% inhibition. To evaluate the prevention efficacy of PTEN gene delivery, N1-S1 cells were infected with adenovirus vectors then implanted into livers of Sprague-Dawley rats to induce Novikoff hepatoma. Injection of PBS- or Ad-GFP-treated N1-S1 cells led to large hepatoma (with an average size of 3-4 cm) with tumor incidence of 80-90%. In contrast, injection of Ad-PTEN-infected N1-S1 cells only induced one hepatoma (with size of 0.1 cm) in six rats, suggesting that pretreatment with PTEN gene delivery effectively abolished the tumorigenic potential of N1-S1 hepatoma cells in vivo. In summary, these results validate the feasibility of PTEN gene delivery as a new promising therapeutic strategy for the treatment of orthotopic hepatoma in immune-competent rats.

No description available.

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.

The immobilization of surface-modified polyamidoamine (PAMAM) dendrimers on the cell surface introduces a novel approach for efficient and specific cellular uptake of therapeutic-carrying nanoparticles. This cell surface-nanoparticle hybridization event takes place via bioorthogonal copper-free click chemistry between a dibenzocyclooctyne (DBCO) group on the dendrimer surface and azide-capped glycans expressed on the cell membrane through metabolic incorporation of azido sugars. This particular cell-nanoparticle hybridization method can be exploited to deliver a variety of therapeutic, genetic or fluorescent payloads directly into cells. Here, this method was employed to deliver plasmid DNA, siRNA and the hydrophobic anticancer drug Camptothecin (CPT) to enhance transfection and therapeutic efficacy. Native, acetylated, and PEGylated generation 4 (G4) PAMAM dendrimers were conjugated with DBCO. When introduced to azide expressing NIH3T3 fibroblasts and HN12 cancer cells, successful surface hybridization was achieved. The physiochemical properties of PAMAM dendrimers allowed for successful hydrophobic drug encapsulation and electrostatic nucleic acid condensation.

No description available.

The delivery of nucleic acids remains the major obstacle for nucleic acid-based therapies such as gene therapy and gene silencing therapies based on RNA interference. In this dissertation we have developed and studied nucleic acid delivery vehicles based on cationic diblock glycopolymers that contain glucosamine and trehalosamine. Practical procedures were developed to synthesize 2-methacrylamido-2-deoxy glucose and 6-methacrylamido-6-deoxy trehalose starting with commercially available carbohydrates and utilizing trimethylsilyl protecting group chemistry. These monomers were polymerized via reversible addition-fragmentation chain transfer (RAFT) polymerization to yield glycopolymers with the desired lengths and low polydispersity indexes. Glycopolymers were chain-extended with aminoethylmethacrylamide to produce cationic diblock copolymers. The ability of cationic diblock copolymers to bind nucleic acids was demonstrated with gel electrophoresis and heparin exclusion assays. Complexes of the synthesized polymers with nucleic acids were studied with dynamic light scattering to reveal nanoparticles of 100-250 nm that were stable in the presence of serum proteins. Quartz crystal microbalance experiments showed that serum proteins adsorb on polytrehalose coated gold surfaces and it was suggested that these interactions may help mask the polytrehalose coated nanoparticles from potential actions of the immune system. Polytrehalose was also shown to suppress water crystallization similarly to trehalose by lowering the energies associated with the water/ice phase transition. The property was utilized to freeze-dry siRNA containing polyplexes which could be re-dissolved in water after lyophilization to yield nanoparticles. The polyplexes formulated with cationic diblock copolymers were shown to efficiently enter cervical cancer cells (HeLa cell line) and glioblastoma cells (U-87 cell line) and to deliver their nucleic acid cargo. Polyglucose-containing polymers were efficient mediators of exogenous gene expression in HeLa cells, and polytrehalose- containing polymers were effective in promoting the target gene down-regulation via RNA interference by delivered siRNA. / Ph. D.