Nanocellulose Aerogels as a Solid Template to Induce Protein Aggregation and Amyloid Formation

For decades, amyloid proteins were mostly studied in the context of their roles as contributors to disease. However, the increasing interest in the development of sustainable materials led to researchers exploiting and engineering amyloids for a myriad of functions. These include filtering heavy metals [1], antimicrobial activity [2], and enhancing biocompatibility [3] to name a few.<br/>The formation of amyloids (ordered aggregates) as opposed to disordered aggregates upon protein unfolding is influenced by various factors such as temperature, pH and interfaces. In this research, we focus on the role of interfaces, particularly we are investigating how solid surfaces influence protein aggregation and amyloid formation. Previously, inorganic solid nanoparticles such as silica nanoribbons, carbon nanotubes, quantum dots, and cerium oxide nanoparticles were shown to induce amyloid formation in proteins such as human β2-microglobulin and Islet Amyloid Polypeptides [4, 5]. However, inorganic and synthetic solid templates are often not sustainable or biocompatible, limiting their utility and acceptability. Therefore, we instead used cellulose, the most abundant biopolymer on earth. Cellulose, obtained from plants, can be converted into cellulose nanofibers with a high surface to volume ratio and cross-linked to form CNF aerogels [6]. In order to induce amyloid formation on their surface, these CNF aerogels were exposed to a solution of hen egg white lysozymes at varying pH. The surface aggregation was studied via various microscopic and spectroscopic methods including FTIR, TIRF, and SEM. Previous reports indicated the adsorption of lysozymes onto the surface of aerogels via electrostatic complexation [7]. Our results indicate not only adsorption of lysozymes, but also the induction of protein aggregation and amyloid formation on the aerogel surface. Additionally, bulk aggregation (or a lack thereof) was investigated using DLS and AFM. By tuning the experimental conditions, amyloid formation was found to either take place exclusively at the aerogel surface or alternatively the presence of aerogels could accelerate bulk aggregation under conditions where little or no lysozyme aggregation was previously reported [2].<br/>Our research provides a new, sustainable method for amyloid aggregation on solid templates. Additionally, the structural integrity of CNF aerogels along with the abundant availability of cellulose provides fascinating new opportunities to create amyloid-cellulose biohybrids.<br/><br/><b>References:</b><br/>1. Soon, W.L., et al., <i>Plant-based amyloids from food waste for removal of heavy metals from contaminated water.</i> Chemical Engineering Journal, 2022. <b>445</b>: p. 136513.<br/>2. Kummer, N., et al., <i>Self-Assembly Pathways and Antimicrobial Properties of Lysozyme in Different Aggregation States.</i> Biomacromolecules, 2021.<br/>3. Janssen, M.I., et al., <i>Coating with genetic engineered hydrophobin promotes growth of fibroblasts on a hydrophobic solid.</i> Biomaterials, 2002. <b>23</b>(24): p. 4847-54.<br/>4. Linse, S., et al., <i>Nucleation of protein fibrillation by nanoparticles.</i> Proceedings of the National Academy of Sciences, 2007. <b>104</b>(21): p. 8691-8696.<br/>5. Pilkington, E.H., et al., <i>Star Polymers Reduce Islet Amyloid Polypeptide Toxicity via Accelerated Amyloid Aggregation.</i> Biomacromolecules, 2017. <b>18</b>(12): p. 4249-4260.<br/>6. Wu, T.T., et al., <i>Dual-porous cellulose nanofibril aerogels via modular drying and cross-linking.</i> Nanoscale, 2020. <b>12</b>(13): p. 7383-7394.<br/>7. Severini, L., et al., <i>Biohybrid Nanocellulose–Lysozyme Amyloid Aerogels via Electrostatic Complexation.</i> ACS Omega, 2022. <b>7</b>(1): p. 578-586.