Characterization, Production, and Applications of Class II Hydrophobins in Drug Formulation and Delivery
Sallada, Nathanael, Biomedical Engineering - School of Engineering and Applied Science, University of Virginia
With an estimated 70% of drugs in early development falling into categories associated with low solubility in aqueous media, and this percentage expected to grow, developing new biocompatible, scalable, and tunable materials to enhance formulation and delivery of poorly soluble drugs is increasingly important. Hydrophobins are small, highly surface-active fungal proteins with broad potential for interface engineering, and show promise in low-solubility drug delivery applications. In this dissertation I characterize fundamental differences in structure-function relationships between two prevailing classes of hydrophobins found in nature, demonstrating that, in contrast to class I hydrophobins, conserved disulfide bonds in our model class II hydrophobin are required for protein structural stability, surface activity at both liquid–liquid and solid–liquid interfaces, and solution self-assembly. With these key structural features in mind, I develop a molecular engineering strategy to overcome a limit in recombinant production using a heterologous yeast host. I show that low yield recombinant strains can be readily engineered to dramatically increase yield up to 30-fold, without further optimization, by synergistic action of increasing target gene copy number and overexpressing relevant chaperone proteins involved in hydrophobic domain stabilization and disulfide bond oxidation. Lastly, I evaluate the engineered class II hydrophobin as a crystallization inhibitor in supersaturated solutions of a model hydrophobic drug compound, flufenamic acid, compared to best performing industry polymers commonly used in amorphous drug delivery systems. I demonstrate that the class II hydrophobin outperforms all competitor polymers, efficiently functioning at much lower required concentrations, and mechanistically functions as an antinucleating agent through two regimes, depending on if the drug concentration exceeds its crystalline or amorphous solubility. Collectively, this dissertation lays groundwork for understanding fundamental class II hydrophobin structure-function relationships, producing recombinant class II hydrophobins at large scale, and applying class II hydrophobins to new hydrophobic drug delivery modalities.
PHD (Doctor of Philosophy)