Shaul Lapidot

Nanocellulose, a tiny fiber with huge applications

Nanocellulose is of increasing interest for a range of applications relevant to the fields of material science and biomedical engineering due to its renewable nature, anisotropic shape, excellent mechanical properties, good biocompatibility, tailorable surface chemistry, and interesting optical properties. We discuss the main areas of nanocellulose research: photonics, films and foams, surface modifications, nanocomposites, and medical devices. These tiny nanocellulose fibers have huge potential in many applications, from flexible optoelectronics to scaffolds for tissue regeneration. We hope to impart the readers with some of the excitement that currently surrounds nanocellulose research, which arises from the green nature of the particles, their fascinating physical and chemical properties, and the diversity of applications that can be impacted by this material.

Expression, cross-linking, and characterization of recombinant chitin binding resilin.

Resilin is a polymeric rubber-like protein secreted by insects to specialized cuticle regions, in areas where high resilience and low stiffness are required. Resilin binds to the cuticle polysaccharide chitin via a chitin binding domain and is further polymerized through oxidation of the tyrosine residues resulting in the formation of dityrosine bridges and assembly of a high-performance protein-carbohydrate composite material. We describe for the first time a comprehensive study on the mechanical, structural and biochemical function of chitin binding recombinant Drosophila melanogaster resilin. Various resilin constructs were cloned including the full length gene enabling Ni-NTA purification, as well as heat and salt precipitation for rapid and efficient purification. The binding isotherms and constants (Kd, Bmax) of resilin to chitin via its chitin binding domain were determined and displayed high affinity to chitin, implying its important role in the assembly of the resilin-chitin composite. The structural and elastic properties were investigated using Fourier Transform Infrared Spectroscopy (FTIR), Circular Dichroism (CD) and Atomic Force Microscopy (AFM) with peroxidase crosslinked solid resilin materials. Generally, little structural organization was found by these biophysical methods, suggesting structural order was not induced by the dityrosine crosslinks. Further, the elastomeric properties found from the full length protein compared favorably with the shorter resilin generated previously from exon 1. The unusual elastomeric behavior of this protein suggests possible utility in biomaterials applications.

Recombinant exon-encoded resilins for elastomeric biomaterials

Resilin is an elastomeric protein found in specialized regions of the cuticle of most insects, providing outstanding material properties including high resilience and fatigue lifetime for insect flight and jumping needs. Two exons (1 and 3) from the resilin gene in Drosophila melanogaster were cloned and the encoded proteins expressed as soluble products in Escherichia coli. A heat and salt precipitation method was used for efficient purification of the recombinant proteins. The proteins were solution cast from water and formed into rubber-like biomaterials via horseradish peroxidase-mediated cross-linking. Comparative studies of the two proteins expressed from the two different exons were investigated by Fourier Transform Infrared Spectroscopy (FTIR) and Circular Dichrosim (CD) for structural features. Little structural organization was found, suggesting structural order was not induced by the enzyme-mediated di-tyrosine cross-links. Atomic Force Microscopy (AFM) was used to study the elastomeric properties of the uncross-linked and cross-linked proteins. The protein from exon 1 exhibited 90% resilience in comparison to 63% for the protein from exon 3, and therefore may be the more critical domain for functional materials to mimic native resilin. Further, the cross-linking of the recombinant exon 1 via the citrate-modified photo-Fenton reaction was explored as an alternative di-tyrosine mediated polymerization method and resulted in both highly elastic and adhesive materials. The citrate-modified photo-Fenton system may be suitable for in vivo applications of resilin biomaterials.

Highly Modified Cellulose Nanocrystals and Formation of Epoxy-Nanocrystalline Cellulose (CNC) Nanocomposites

This work presents an environmentally friendly, iodine-catalyzed chemical modification method to generate highly hydrophobic, optically active nanocrystalline cellulose (CNC). The high degree of ester substitution (DS = 2.18), hydrophobicity, crystalline behavior, and optical activity of the generated acetylated CNC (Ac-CNC) were quantified by TEM, FTIR, solid 13C NMR, contact angle, XRD, and POM analyses. Ac-CNC possesses substantial enhancement in thermal stability (16.8%) and forms thin films with an interlayer distance of 50-150 nm, presenting cavities suitable for entrapping nano- and microparticles. Generated Ac-CNC proved to be an effective reinforcing agent in hydrophobic polymer matrices for fabricating high performance nanocomposites. When integrated at a very low weight percentage (0.5%) in an epoxy matrix, Ac-CNC provided for a 73% increase in tensile strength and a 98% increase in modulus, demonstrating its remarkable reinforcing potential and effective stress transfer behavior. The method of modification and the unique properties of the modified CNC (hydrophobicity, crystallinity, reinforcing ability, and optical activity) render them a novel bionanomaterial for a range of multipurpose applications.

Multifunctional Cellulosic Scaffolds from Modified Cellulose Nanocrystals

A biobased cellulosic scaffold material was made through freeze-drying ice-templating of functionalized cellulosic nanomaterials. The resulting interconnected highly porous scaffold was primarily composed of highly esterified, strong network of ultrathin cellulosic layers. The prepared cellulosic scaffold material displayed multifunctional properties of hydrophobicity, oleophilicity and lipophilicity, which could selectively absorb milkfat, hydrophobic proteins, various organic solvents and oils. Diverse potential for the structural and medical applications, such as tissue engineering, drug delivery, and oil and fat accumulation are proposed.

Spider Silk-CBD-Cellulose Nanocrystal Composites: Mechanism of Assembly.

The fabrication of cellulose-spider silk bio-nanocomposites comprised of cellulose nanocrystals (CNCs) and recombinant spider silk protein fused to a cellulose binding domain (CBD) is described. Silk-CBD successfully binds cellulose, and unlike recombinant silk alone, silk-CBD self-assembles into microfibrils even in the absence of CNCs. Silk-CBD-CNC composite sponges and films show changes in internal structure and CNC alignment related to the addition of silk-CBD. The silk-CBD sponges exhibit improved thermal and structural characteristics in comparison to control recombinant spider silk sponges. The glass transition temperature (Tg) of the silk-CBD sponge was higher than the control silk sponge and similar to native dragline spider silk fibers. Gel filtration analysis, dynamic light scattering (DLS), small angle X-ray scattering (SAXS) and cryo-transmission electron microscopy (TEM) indicated that silk-CBD, but not the recombinant silk control, formed a nematic liquid crystalline phase similar to that observed in native spider silk during the silk spinning process. Silk-CBD microfibrils spontaneously formed in solution upon ultrasonication. We suggest a model for silk-CBD assembly that implicates CBD in the central role of driving the dimerization of spider silk monomers, a process essential to the molecular assembly of spider-silk nanofibers and silk-CNC composites.

Expression, cross-linking and characterization of recombinant chitin binding resilin

Resilin is a polymeric rubber-like protein secreted by insects to specialized cuticle regions, in areas where high resilience and low stiffness are required. Resilin binds to the cuticle polysaccharide chitin via a chitin binding domain and is further polymerized through oxidation of the tyrosine residues resulting in the formation of dityrosine bridges and assembly of a high-performance protein-carbohydrate composite material. We describe for the first time a comprehensive study on the mechanical, structural and biochemical function of chitin binding recombinant Drosophila melanogaster resilin. Various resilin constructs were cloned including the full length gene enabling Ni-NTA purification, as well as heat and salt precipitation for rapid and efficient purification. The binding isotherms and constants (K d , B max ) of resilin to chitin via its chitin binding domain were determined and displayed high affinity to chitin, implying its important role in the assembly of the resilin-chitin composite. The structural and elastic properties were investigated using Fourier Transform Infrared Spectroscopy (FTIR), Circular Dichroism (CD) and Atomic Force Microscopy (AFM) with peroxidase crosslinked solid resilin materials. Generally, little structural organization was found by these biophysical methods, suggesting structural order was not induced by the dityrosine crosslinks. Further, the elastomeric properties found from the full length protein compared favorably with the shorter resilin generated previously from exon 1. The unusual elastomeric behavior of this protein suggests possible utility in biomaterials applications.