The delivery of biomolecules by cell penetrating peptides (CPPs) is an innovative therapeutic strategy. However delivery efficiency is hindered by the entrapment of CPPs in vesicles, degradation, or recycling out of cells, which limits their delivery into the cell cytoplasm and nucleus. To overcome these barriers, we investigated a bacterial protein domain derived from Pseudomonas aeruginosa, Exotoxin A (ETA, residues 253-412) that is able to exit vesicular compartments. A series of CPP-ETA(253-412) fusion proteins were constructed, expressed, and purified. Confocal microscopy and flow cytometry confirmed the internalization at 37oC of constructs containing CPPs (poly-arginine or TAT). In addition, constructs containing CPP-ETA(253-412)-eGFP were shown to relocate from endosomes to the cytosol. CPP-ETA(253-412) constructs were also able to act as carriers of DNA cargos facilitating their delivery to the cytosol. The ETA(253-412) translocation domain may prove useful for the intracellular delivery of drugs, protein therapeutics, siRNA delivery, and vaccine formulations.

The delivery of biomolecules by cell penetrating peptides (CPPs) is an innovative therapeutic strategy. However delivery efficiency is hindered by the entrapment of CPPs in vesicles, degradation, or recycling out of cells, which limits their delivery into the cell cytoplasm and nucleus. To overcome these barriers, we investigated a bacterial protein domain derived from Pseudomonas aeruginosa, Exotoxin A (ETA, residues 253-412) that is able to exit vesicular compartments. A series of CPP-ETA(253-412) fusion proteins were constructed, expressed, and purified. Confocal microscopy and flow cytometry confirmed the internalization at 37oC of constructs containing CPPs (poly-arginine or TAT). In addition, constructs containing CPP-ETA(253-412)-eGFP were shown to relocate from endosomes to the cytosol. CPP-ETA(253-412) constructs were also able to act as carriers of DNA cargos facilitating their delivery to the cytosol. The ETA(253-412) translocation domain may prove useful for the intracellular delivery of drugs, protein therapeutics, siRNA delivery, and vaccine formulations.

Applications of nanotechnology in medicine, also known as nanomedicine, is a rapidly growing field as it holds great potential in the development of novel therapeutics toward treatment of various diseases. Shell crosslinked knedel-like nanoparticles (SCKs) that are self assembled from amphiphilic block copolymers into polymeric micelles followed by crosslinking selectively throughout the shell domain have been investigated as theranostic agents for the delivery of nucleic acids and incorporation of imaging probes. The main focus of this dissertation is to design and develop unique multifunctional bio-synthetic hybrid nanoparticles that can carry agents for radiolabeling, moieties for inducing stealth properties to minimize protein adsorption in vivo, ligands for site-specific targeting, therapeutic payloads, and are optimized for efficient delivery of cargoes intracellularly and to the target sites toward constructing novel nanoscopic objects for therapy and diagnosis. Alteration of polymeric building blocks of the nanoparticles provides opportunities for precise control over the sizes, shapes, compositions, structures and properties of the nanoparticles. To ensure ideal performance of nanoparticles as theranostic agents, it is critical to ensure high intracellular bioavailability of the therapeutic payload conjugated to nanoparticles. Special efforts were made by employing well-defined multi-step polymerization and polymer modification reactions that involved conjugation of peptide nucleic acids (PNAs) to chain terminus of poly(ethylene glycol) (PEG) chain grafts such that they were presented at the outermost surface of SCKs. Additionally, chemical modification reactions were performed on the polymer backbone to integrate positive charges onto the shell of the nanoparticles to afford cationic SCKs (cSCKs) for facilitating cellular entry and electrostatic interactions with negatively charged nucleic acids. Covalent conjugation of F3, a tumor homing peptide, post-assembly of the nanoparticles enhanced cellular uptake and knockdown of nucleolin (a shuttling protein overexpressed at the sites of angiogenesis) and thus inhibiting tumor cell growth. Furthermore, these polymer precursors of the cSCKs were modified with partial to full incorporation of histamines to facilitate their endosomal escape for efficient delivery into the cytosol. The cSCKs were further templated onto high aspect ratio anionic cylinders to form hierarchically-assembled nanostructures that bring together individual components with unique functions, such as one carrying a therapeutic payload and the other with sites for radiolabeling. These higher order nanoobjects enhance circulation in vivo, have capabilities to package nucleic acids electrostatically and contain sites for radiolabeling, providing an overall advantage over the individual components, which could each facilitate only one or the other of the combined functions. Hierarchically-assembled nanostructures were investigated for their cellular uptake, transfection behavior and radiolabeling efficiency, as the next generation of theranostic agents.

Liposomal doxorubicin is a clinically important drug formulation indicated for the treatment of several different forms of cancer. For doxorubicin to exert a therapeutic effect, it must gain access to the nucleus. However, a large proportion of the liposomal doxorubicin dose fails to work because it is sequestered within endolysosomal organelles following endocytosis of the liposomes due to the phenomenon of ion trapping. Listeriolysin O (LLO) is a pore-forming protein that can provide a mechanism for endosomal escape. The present study demonstrates that liposomal coencapsulation of doxorubicin with LLO enables a significantly larger percentage of the dose to colocalize with the nucleus compared to liposomes containing doxorubicin alone. The change in intracellular distribution resulted in a significantly more potent formulation of liposomal doxorubicin as demonstrated in both the ovarian carcinoma cell line A2780 and its doxorubicin-resistant derivative A2780ADR.

Doctor of Philosophy / Department of Diagnostic Medicine and Pathobiology / Kyeong-Ok Chang / Caliciviruses are important pathogens of humans and animals. Noroviruses are major causes of foodborne gastroenteritis cases, but their research is hindered due to the inability to grow human noroviruses in cell culture. Detailed studies on entry events of caliciviruses are lacking and may be crucial for development of cell culture models. We examined the entry events of caliciviruses using porcine enteric calicivirus (PEC), feline calicivirus (FCV) and murine norovirus-1 (MNV-1). PEC replication in LLC-PK cells requires bile acid in the medium, but the mechanism is not well understood. Our studies showed that bile acids are required in the early stage of virus replication, and while internalization of PEC is not dependent of them, they are required for endosomal escape and successful replication. Further examination on virus entry, we demonstrated that endosomal acidification and cathepsin L activity are essential in the replication of PEC, FCV and MNV-1. The results showed that inhibition of endosomal acidification or cathepsin L activity led to retention of viruses in the endosomes. Also we demonstrated that recombinant cathepsin L cleaved structural protein of PEC, FCV or MNV-1, which suggests that the enzyme may facilitate uncoating viruses in endosomes. In addition to bile acids, we found that a cold shock treatment during virus entry supported PEC replication by facilitating the endosomal escape. While PEC alone did not induce ceramide formation, bile acids or cold shock treatment induce ceramide formation on endosomes through activation acid sphingomyelinase (ASM), and this event was crucial for virus replication because inhibition of ASM blocked ceramide formation and significantly reduced PEC replication. Incubation of FCV or MNV-1 with cells led to ceramide formation during virus entry, and inhibition of ASM also significantly reduced their replication. Inhibition of ASM led to endosomal retention of PEC, FCV or MNV-1 during virus entry, which may be the reason for the reduction of viral replication. These studies revealed the important and common events during calicivirus entry for successful replication, including virus endosomal escape, cathepsin L activity and ASM/ceramide formation. This detailed information may provide clues for understanding the replication of fastidious caliciviruses and for potential therapeutic targets.

The ability of cell-penetrating peptides to cross plasma membranes has been explored for various applications, including the delivery of bioactive molecules to inhibit disease-causing cellular processes. The uptake mechanisms by which cell-penetrating peptides enter cells depend on the conditions, such as the cell line the concentration and the temperature. To be used as therapeutics, each novel cell-penetrating peptide needs to be fully characterized, including their physicochemical properties, their biological activity and their uptake mechanism. Our group has developed a series of highly performing, non-toxic cell-penetrating peptides, all derived from the original sequence of transportan 10. These analogs are called PepFects and NickFects and they are now a diverse family of N-terminally stearylated peptides. These peptides are known to form noncovalent, nano-sized complexes with diverse oligonucleotide cargoes. One bottleneck that limits the use of this technology for gene therapy applications is the efficient release of the internalized complexes from endosomal vesicles. The general purpose of this thesis is to reveal the mechanisms by which our in house designed peptides enter cells and allow the successful transport of biofunctional oligonucleotide cargo. To reach this goal, we used both biophysical and cell biology methods. We used spectroscopy methods, including fluorescence, circular dichroism and dynamic light scattering to reveal the physicochemical properties. Using confocal and transmission electron microscopy we observed and tracked the internalization and intracellular trafficking. Additionally we tested the biological activity in vitro and the cellular toxicity of the delivery systems. We conclude that the transport vectors involved in this study are efficient at perturbing lipid membranes, which correlates with their remarkable capacity to transport oligonucleotides into cells. The improved and distinct capacities to escape from endosomal vesicles can be the result of their different structures and hydrophobicity. These findings extend the knowledge of the variables that condition intracellular Cell-penetrating peptide mediated transport of nucleic acids, which ultimately translates into a small step towards successful non-viral gene therapy.

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<p>miRNAs therapeutics have emerged as a potential cancer therapeutics due to their unique ability to target multiple genes, allowing a single miRNA to act as a multi-drug cocktail. However, toxicity associated with current delivery vehicles as well as the sensitivity of the miRNA to serum nucleases are critical hurdles that stand against their clinical utility. Ligand-targeted delivery approaches such as small molecules, aptamers, antibodies or glycoconjugates provide a promising strategy to achieve specific delivery of the therapeutic cargo to the intended targeted cells without any toxicity. We hypothesized that combining both ligand targeted approach along with modifying the miRNA would provide a perfect delivery system which does not only achieve specific delivery but also reduce the therapeutic dose. The data presented in this dissertation shows (i) that miR-34a delivered using a combination of targeting ligand (DUPA) and an endosomal escape agent (nigericin) enables selective delivery of miR-34a to prostate cancer cells, (ii) that the use of full chemical modification approach enhances miR-34a stability and activity both in vitro and in vivo. Overall, these results provide an advancement in the miRNA delivery field and will help the development of miRNA based therapeutics against cancer.</p>

My thesis concerns spectroscopic studies (NMR, CD and fluorescence) of peptides with functions in biotechnology and biology, and their interactions with a model membrane (large unilamellar phospholipid vesicles). The resorufin-based arsenical hairpin binder (ReAsH) bound to a short peptide is a useful fluorescent tag for genetic labeling of proteins in living cells. A hairpin structure with some resemblance to type II β-turn was determined by NMR structure calculations (Paper I). Cell-penetrating peptides (CPPs) are short (30-35 residues), often rich in basic amino acids such as Arg. They can pass through the cell membrane and deliver bioactive cargoes, making them useful for biotechnical and pharmacological applications. The mechanisms of cellular uptake and membrane translocation are under debate. Understanding the mechanistic aspects of CPPs is the major focus of Papers II, III, and IV. The effect of the pyrenebutyrate (PB) on the cellular uptake, membrane translocation and perturbation of several CPPs from different subgroups was investigated (Paper II). We concluded that both charge and hydrophobicity of the CPP affect the cellular uptake and membrane translocation efficiency. Endosomal escape is a crucial challenge for the CPP applications. We modeled the endosome and endosomal escape for different CPPs to investigate the corresponding molecular mechanisms (Papers III and IV). Hydrophobic CPPs were able to translocate across the model membrane in the presence of a pH gradient, produced by bacteriorhodopsin proton pumping, whereas a smaller effect was observed for hydrophilic CPPs. Dynorphin A (Dyn A) peptide mutations are associated with neurodegenerative disorders, without involvement of the opioid receptors. The non-opioid activities of Dyn A may involve membrane perturbations. Model membrane-perturbations by three Dyn A mutants were investigated (Paper V). The results showed effects to different degrees largely in accordance with their neurotoxic effects. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

No description available.