Improved Stability, Nanomechanical Analysis, and Biomedical Applications of β-Lactoglobulin Fibrils

Proteins have the ability to assemble into long, narrow fibril structures known as amyloid proteins. This can occur under the simple parameters of prolonged heat treatment in acidic conditions. While much of the research surrounding amyloid proteins has been targeted towards their association with neurodegenerative diseases, such as Alzheimer’s, Huntington’s, and Parkinson’s diseases, recent focus has moved to applications of amyloid proteins fabricated from natural commodities, such as whey protein. A significant limitation of these nanostructures is their instability in pH systems near the isoelectric point of the protein, causing aggregation and degradation of the fibril structure that limits their potential applications. This dissertation describes attempts to improve the pH stability of fibrils composed of β-lactoglobulin, a protein from dairy whey, as well as the potential application of these fibrils in composite materials for biotechnology. Chapter 1 provides an overview of protein fibrils along with their analysis using atomic force microscopy. Chapter 2 describes the ability to improve the stability of fibrils through the electrostatic interaction of cationic polymers chitosan and polyethylenimine to the fibril surface. To confirm their stability, turbidity, zeta potential, and atomic force microscopy were used. Extensive AFM image analysis indicated a significant increase in both the persistence length and the contour length of the fibrils at pH values at and above 5. To further investigate potential applications in areas such as composite materials, β-lactoglobulin fibrils were analyzed using a nanomechanical analysis technique known as bimodal force spectroscopy (Chapter 3). The fibrils were found to have a Young’s Modulus of 1.6 GPa. This result was confirmed using PeakForce Quantitiative Nanomechanical Analysis, an established analysis technique that has been used to investigate amyloid proteins. As an example of the versatility of stabilized fibrils as supportive materials, chitosan-coated fibrils were tested for their biocompatibility to support the growth and differentiation of human Mesenchymal Stem Cells (Chapter 4). Cells were seeded onto 2-dimmensional fibril networks and were observed to form multicellular spheroids. Multipotency and osteogenic differentiation capabilities of the cells were maintained up to 7 days of culturing on the fibril surfaces. Upon analysis of the adipogenesis potential, a significant increase was observed for cells plated on chitosan-coated fibrils, showcasing potential applications of stabilized β-lactoglobulin fibrils in biomedicine