Molecular Engineering of Nanocellulose for Biomedical Applications

Cellulose is an abundant biopolymer that can be synthesized by many different organisms from all kingdoms of life with plants offering the highest availability. Cellulose-derived fibrils and particles with dimensions in the nanometer range are referred to as nanocellulose which is a nature-based nanomaterial with interesting- and potentially useful properties. Nanocellulose combines in a unique way the characteristic cellulose properties like the non-toxicity and a high biodegradability with the benefits of nanoscale materials like high surface area and mechanical strength. Understanding the material properties and effective modification strategies are crucial steps to introduce bioactive properties to nanocellulose and thus further facilitate their use in biomedical applications. This thesis provides an in-depth understanding of how active proteins and peptides can be efficiently immobilized to nanocellulose, i.e. wood-derived nanocellulose (NFC), nanocellulose crystals (CNC) and bacterial cellulose (BC), and how control over the bioactivity of the resulting biocomposite material can be achieved. Different biochemical modification strategies based on physical, covalent, or bioaffinity immobilization procedures are explored and rationally designed using proteins with different biochemical and structural features. Moreover, the question of how the intricate mechanism of immobilization and modification affects both the biomolecules activity and, the physical properties of the nanocellulose substrate is answered. By studying the physical adsorptive properties of TEMPO-oxidized nanofibrillated cellulose (TONFC) as a first proof-of-principle, it could be demonstrated that different biomolecules can be immobilized via tunable electrostatic interactions to the charged TONFC (Chapter 2). Moreover, a bottom-up functionalization strategy was reported and combined a spontaneous and tunable physical interaction and stable covalent bonding. Following this strategy, it was possible to produce a highly active TONFC-protease biocomposite material with increased enzymatic activity as compared to the soluble enzyme form. The described bottom-up functionalization strategy could be further improved by optimizing the covalent coupling step to immobilize a sensing protein (Chapter 3). A bio-hybrid copper-sensitive nanocellulose paper film was thus developed based on the red-fluorescent protein C-Phycocyanin (CPC). Although immobilization and covalent coupling led to changes in the copper sensitivity of CPC, the protein stability was significantly enhanced. In addition, the sensing paper films were demonstrated to be reusable for several cycles over several days with a maximal sensing performance in the nanomolar range and activity for example in human serum. A closer look on the physical interactions of biomolecules and TONFC in more detail, pronounced effects on both the bioactivity and the material properties could be elucidated (Chapter 4). When the antimicrobial peptide (AMP) nisin was physically immobilized to TONFC, the fibril surface properties changed, undergoing unravelling and stiffening as could be elucidated by small angle X-ray scattering combined with model based data analysis. The effect was even more pronounced at basic pH. Compared to the nisin molecule in solution, the TONFC-nisin bio-nanocomposite suspension showed a marked enhanced antibacterial action against different bacterial strains, including a pathogenic S. aureus. The increased bioactivity observed, was not restricted to the enzyme Papain and nisin. By engineering a second AMP tet009 to carry either a cellulose binding peptide (CBP) or cellulose binding protein motif (CBM), a one-step spontaneous immobilization on pristine cellulose of plant and bacterial origin, as fibrils or crystals, was achieved (Chapter 5). The interaction of tet009 with CBP and NFC or CNC significantly enhanced the antimicrobial activity of tet009 against both Gram-negative and Gram-positive pathogenic strains. These observations demonstrated that a bioaffinity-based functionalization strategy could be applied to different cellulose substrates without requiring the previous modification of cellulose. Furthermore, the absence of acute cytotoxicity of the plant and bacterial cellulose preparations used in this study was assessed and a procedure to reduce the inflammatory contaminants possibly present in nanocellulose preparations was developed. Endotoxin levels were thus reduced to levels acceptable for further in vitro and in vivo testing. Through this work, biochemical surface modification strategies of nanocellulose were established and demonstrated to be feasible for the future development of functional nanocellulose-based biocomposite material concepts as enzyme carriers, point-of-care biosensors delivery vehicles for antimicrobial drugs. Besides, the absence of acute cytotoxicity and the low level of endotoxin contamination achieved for NFC and BC films are promising for a further systematic analysis of the immune response towards these materials and their future implementation in functional coatings of implants, wound dressings or other biomedical applications