Date of Award

Spring 5-15-2015

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Computational & Molecular Biophysics)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

The development of small molecules as therapeutic agents for targeted disease treatment is unable to keep up with the rapid expansion of databases cataloguing disease-causing proteins enabled by high throughput analysis of patient samples. As an alternative, the use of small interfering RNA (siRNA) provides a simple means of efficiently and specifically silencing the expression of these pathogenic proteins without the need for screening and development required of small molecules. Unfortunately, the delivery of siRNA is not trivial, and existing technology is characterized by either poor siRNA transfection efficiency or induction of cytotoxicity. Detailed work by many groups has supported the finding that low transfection efficiency is often attributable to endosomal entrapment. This phenomenon prevents siRNA from accessing the cytoplasmic compartment where it is active. Consequently, development of new siRNA vectors is required to safely promote endosomal escape and delivery of siRNA to the cytoplasm. This work focuses on the development and characterization of peptides derived from melittin, the membrane-lytic component of honeybee venom, for siRNA delivery. The most active melittin derivative, p5RHH, includes modifications to decrease cytotoxicity, increase siRNA binding, and allow triggered siRNA release in response to the acidic environment encountered during endocytosis. These peptides bind siRNA to form nanoparticles of 50 to 200 nm in diameter with a positive zeta potential (+12 mV). This low magnitude surface charge cannot stabilize the particles against flocculation, necessitating a subsequent coating with serum albumin to achieve a stable formulation. p5RHH-mediated transfection is characterized by an IC50 in the range of 25 to 100 nM without cytotoxicity at all tested doses. Furthermore, the activity of p5RHH is attributed to efficient endocytosis via macropinocytosis with nanoparticle disassembly in the endosome. Particle disassembly releases both siRNA and peptide, allowing p5RHH to disrupt the endosomal membrane and siRNA to access the cytoplasmic compartment. To demonstrate broad transfection potential, p5RHH-mediated transfection has been utilized to treat in vitro models of cancer, angiogenesis, and atherosclerosis. Furthermore, in vivo studies demonstrate the ability of p5RHH/siRNA nanoparticles to deposit in tumors with little accumulation in clearance organs such as the liver and spleen. Instead, these studies reveal siRNA clearance via the kidney. Our results indicate that melittin can be modified for efficient and safe nanoparticle-mediated siRNA transfection, potentially enabling the clinical use of siRNA. Moreover, our analysis of p5RHH's mechanism of action provides a framework to guide the future development of peptide vectors for siRNA transfection.

Language

English (en)

Chair and Committee

Samuel Wickline

Committee Members

Elliot Elson, Kathleen Hall, Phyllis Hanson, Katherine Henzler-Wildman, Paul Schlesinger

Comments

Permanent URL: https://doi.org/10.7936/K7MP51FV

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Biology Commons

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