Self-assembled Antimicrobial Amyloid-Nanocellulose Biohybrids
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2023
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Doctoral Thesis
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Abstract
Amyloids derived from low-cost food proteins and nanocellulose from wood pulp are renewable and biodegradable materials with a wide range of potential applications, including water purification, biomaterials, and bioplastics. When amyloids are made from hen egg white lysozyme (HEWL), they exhibit a non-catalytic broad-spectrum antimicrobial activity based on their positive charge interacting with the negatively charged cell walls and cell membranes of microorganisms. Using disulfide bond reduction to induce self-assembly, colloidally stable amyloid-like, worm-shaped HEWL aggregates (worms) were produced and compared to amyloid fibrils made via an acid hydrolysis pathway. The aggregation kinetics of the HEWL worms was strongly pH-dependent, due to reduced double layer repulsion at higher pH, leading to large aggregates and sedimentation.
The aggregation at pH 4.5 led to 1-dimensional worms and induced a structural transition from α-helices to β-sheets. Both HEWL worms and amyloid fibrils showed broad-spectrum antimicrobial activity against Staphylococcus aureus, Escherichia coli, and Candida albicans. Amyloid fibrils exhibit the strongest antimicrobial effect, due to their increased positive charge compared to native HEWL leading to stronger interactions with the negatively charged cell walls and cell membranes of the microorganisms and cell lysis. The HEWL amyloid fibrils were coated onto TEMPO-oxidized cellulose nanofibril (TO-CNF) nanopapers using a coating mechanism based on the formation of 2D foam films. Exploiting the surface activity of proteins, a 2D foam film was pulled out of a HEWL amyloid fibril suspension using a loop, similar to the one used when blowing bubbles.
To enhance the stability of the foam film, different concentrations and pH values of the amyloid suspension were tested. The foam stability was increased drastically at pH 6, V due to the deprotonation of the acidic amino acids, introducing negative charges and reducing the adsorption barrier caused by double layer repulsion. Using ultrafiltration, amyloid fibrils (40% of the protein mass) were separated from unconverted peptides (60%) and tested for their foam stability. It turned out that the unconverted peptides were the main species stabilizing the air-water interface, rather than the amyloid fibrils themselves. By depositing the 2D foam film onto various substrates, a uniform coating layer with a thickness of 30 nm was formed.
This thickness was sufficient to produce a positively charged HEWL amyloid coating on negatively charged TO-CNF nanopapers. The coating showed a similar trend in terms of antimicrobial effect against the same pathogens (S. aureus, E. coli and C. albicans) tested before with the amyloid suspensions. However, the charge-mediated antimicrobial effect was not as strong due to the loss of surface area of the amyloid fibrils compressed into the coating layer. Overall, the foam film coating offers a facile method for the functionalization of TO-CNF nanopapers with antimicrobial HEWL amyloids, which could potentially be exploited for the production of wound dressing materials. In order to enhance the mechanical properties of hybrid hydrogels, the colloidal inter-actions between amyloid fibrils and nanocellulose and their tunability was studied. Polyelectrolyte complexation was dominating the interactions between positively charged HEWL amyloids and negatively charged cellulose nanocrystals (CNCs), while negatively charged β-lactoglobulin (BLG) amyloids could be co-dispersed with CNCs. The complexation increased the elasticity of the amyloid network by cross-linking individual fibrils. Therefore, concentration-dependent rheological measurements were per-formed to fit elasticity-concentration scaling laws. The scaling of the elasticity with amyloid concentration was in good agreement with results from literature and theoretical models.
The nanocellulose morphology contributes differently to elasticity: CNCs VI cause amyloid bundling, while TO-CNFs add to a second network. Finally, a pH-mediated self-assembly approach was used to create homogeneous hydrogels made from HEWL and BLG amyloids in combination with nanocellulose.
The mixing was done above the isoelectric point of the proteins, so they would be negatively charged and make stable co-dispersions with nanocellulose. Dialysis was used to reduce the pH to cross the isoelectric point, introducing positive charges on the amyloids and causing polyelectrolyte complexation and gelation. The type of nanocellulose morphology (CNCs or CNFs) did not significantly affect the elastic modulus in shear and com-pression tests, but hybrid gels with TO-CNFs displayed a higher dynamic yield point, enabling them to withstand greater deformations. These findings promote the under-standing of amyloid fibril and nanocellulose interactions and offer valuable strategies for optimizing hybrid material assembly. Overall, this thesis provides important insights on the self-assembly of HEWL aggregates and their co-assembly with nanocellulose. Due to the high sensitivity of proteins to pH changes, tuning the protonation of amyloid fibrils is the best way to control their self-assembly. With the knowledge gained from this thesis, the design of new hybrid materials based on amyloid fibrils and nanocellulose can be developed.
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Examiner : Fischer, Peter Alfons
Examiner : Nyström, Gustav
Examiner: Isa, Lucio
Examiner : Thielemans, Wim
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ETH Zurich
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08821 - Fischer, Peter (Tit.-Prof.)