Date of Award

2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Chemical and Biomolecular Engineering

First Advisor

Daniel A. Hammer

Abstract

Drug carrier plays a critical role in achieving therapeutic effect in human or animals. Existing commercial drug carriers are mostly chemically synthesized, making the materials polydisperse in size, carry heavy residuals and extremely difficult to functionalize for bioactivity. Recombinant protein expressed through molecular biology presents a promising alternative to chemically synthesized materials due to several advantages. Molecular biology techniques allow for precise design of the genetic sequence, thus giving the recombinant protein the ability to incorporate specific motifs that mediates cell recognition. Protein expressed through molecular biology would have the exact amino acid sequence as designed and monodisperse in weight, which makes recombinant protein superior than synthesized materials for quality control purposes. For this thesis, we have chosen a naturally occurring plant protein oleosin, which has been previously demonstrated in our lab to self-assemble into a variety of nanostructures upon gene modification, as the starting template. We have designed many variants of oleosin to self-assemble into spherical micelles and carry bioactive motifs. First, we created a variant of oleosin that possesses dual control by protecting the bioactive ligand RGDS with a thrombin cleavable domain. Cellular uptake studies showed that this model is effective in protecting 74% of the RGDS bioactivity in 15 hours when interacted with breast cancer cells. We further enhanced the cellular uptake performance of oleosin by inserting a synergy PHSRN and cell penetrating peptide HIV-1 Tat, where the Tat peptide and the RGDS motif combination increased cell uptake by sixfold in 15 hours. We proved the ability of oleosin micelles to carry a cargo by successfully encapsulating an anti-cancer drug paclitaxel into oleosin micelles through brief sonication, and achieved integrin mediated cell killing on breast cancer cells over 15 hours. We then applied our oleosin variants to other cell lines by functionalizing with AFA and BPT ligands, and observed significantly improved cell killing on non-small cell lung cancer cells. All protein variants were confirmed for molecular weight and purity by gel electrophoresis and mass spectroscopy, and the second structures analyzed through circular dichroism. Protein self-assembly sizes were analyzed by dynamic light scattering. Oleosin micelles were stable in aqueous solutions for at least one month under refrigerated conditions, and has been found to be stable after drug encapsulation and variant blending. Oleosin has been shown to be a versatile and powerful tool for drug applications, and these results opened a new horizon for oleosin to be further engineered and utilized for therapeutic applications.

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