Monday, November 24, 2014

Low MW FITC tagged chitosan (KITO-3) from Akina, Inc. used for investigation of cuticle formation.

PolySciTech ( provides a wide array of research polymers including chitosan derivatives. A recent PhD thesis by Patricia Ann Sprouse utilized KITO-3 (low molecular weight chitosan FITC tagged) in order to investigate interactions between carbohydrates and proteins within cuticles. Read more: Sprouse, Patricia Ann. "Evaluation of Structure-Property Relationships within Insect Cuticle to Identify Motifs for Biomaterial Design." (2014).

“ABSTRACT: Biological materials have mechanical properties that have been highly tuned for specific functions. The properties are governed by combinations of molecular interactions that are incorporated at multiple length scales in hierarchical structures. Establishing the links between molecular interactions and physical properties in biological materials has the potential to improve the design of structural biomaterials by offering effective design strategies. Insect cuticle, in particular, is an interesting model for studying structure-property relationships because it can have a wide range of mechanical properties despite a limited compositional makeup. The elytron, or outer wing cover, of the beetle is a strong and tough biomaterial made mostly of proteins, chitin, catechols and water. The objective of this thesis was to investigate molecular interactions among the components of elytral cuticle to both understand their role in governing in vivo properties as well as their future potential as biomaterial design motifs. The objective was addressed using two approaches. First, the intact elytron was studied to identify material design concepts that contribute to the strong and tough physical properties of the structure. Second, simplified systems of cuticular components were examined in vitro to help understand the in vivo roles of specific interactions with the future goal of providing new biomaterial design motifs. Studies on the intact elytron from the yellow mealworm beetle, Tenebrio molitor, determined that the cuticle is not compositionally uniform throughout development. Instead, new material is continuously secreted from the epidermal cells. The color change and the frequency dependence of the cuticle plateaued after 48 hours, indicating that the initially secreted material matures within 48 hours of eclosion. The mature cuticle bears the tensile load while additional material is subsequently deposited and matured over a week of development. Important contributions of both covalent and non-covalent interactions were identified in cuticle using dynamic mechanical analysis over a broad range of frequency, temperature and by treatments with non-covalent bond breaking-solvents. Indirect evidence pointed to the dominant role of covalent interactions in stabilizing the cuticle shortly after secretion from the epidermal cells. Dynamic mechanical analysis showed that the storage modulus of fully tanned and dried elytra was less frequency dependent (n = 0.013 ± 0.004) than the modulus of untanned and dried elytra (n = 0.05 ± 0.024), even at frequencies as low as 0.01 rad/s. Additionally, increasing the temperature from 25 to 150°C, up to the point of degradation, resulted in only a 3.5 fold decrease in the storage modulus and modest rise in the tan δ curve for fully tanned elytra. To further separate the role of covalent and non-covalent interactions in elytral cuticle, solvents were used to disrupt non-covalent interactions. More material could be extracted from partially tanned elytra than from fully tanned elytra, most likely reflecting the lower degree of covalent crosslinking in the immature layers of cuticle within partially tanned elytra. Soaking fully tanned elytra in formic acid, which disrupts hydrogen bonding, caused the static mechanical properties to resemble those of partially tanned elytra, which is consistent with the view that covalent crosslinks within the load bearing layers of cuticle are established within 48 hours. The role of non-covalent interactions was also highlighted by mechanical tests after treating with formic acid. The solvent caused a 5-fold decrease in the Young's modulus of fully tanned elytra, suggesting that a significant portion of the stiffness (which increases 20-fold with maturation) is provided by non-covalent interactions. The added stiffness provided by non-covalent interactions appears to have been provided without compromising toughness; that is, apparent hydrogen bonding caused a proportional increase in fracture stress and decrease in fracture strain, thus leaving toughness unchanged. Despite uncovering important contributions from covalent and non-covalent interactions within the network as a whole, the complexity of the biological structure makes it difficult to identify the exact interactions contributing to the physical properties. Thus, simplified systems were used to study specific molecular interactions in vitro. The hypothesized interactions that were studied in vitro, namely protein-chitin complexation, protein-catechol and protein-protein crosslinks, and protein-Ni2+ complexation, were studied using two abundant cuticle proteins in the elytra of the red flour beetle, Tribolium castaneum, CPR27 and CP30. CPR27 has a conserved sequence of amino acids that has been hypothesized to bind chitin. Results from active microrheology showed that addition of CPR27 to fluorescently-labeled aqueous chitosan solutions caused a 2-fold decrease in viscosity. Since this microrheology technique allows for the simultaneous visualization of microstructure, the appearance of bright fluorescent spots was observed along with the drop in viscosity. Together the results indicated that CPR27 complexation with chitosan caused aggregation that effectively removed material from the vicinity of the microrheology probes. CP30, which does not contain the conserved chitin-binding sequence, did not display similar evidence of complexation. The role of quinone crosslinking of both proteins with the catechol, N-&beta-alanyldopamine, was examined using passive microrheology. Microparticle formation was observed in solutions containing protein and laccase, catechol and laccase, and protein, catechol and laccase. However, elasticity was observed only when the protein was present, consistent with the hypothesis that quinone crosslinking has a mechanical role in cuticle, as concluded by the studies on elytra. However, further tests are needed to reproduce the data and verify the origin of elasticity. The highly unusual sequence of highly repetitive short blocks of positive and negative residues in CP30 suggested that it may self-assemble or interact with multivalent metal ions. The protein was found to aggregate or adsorb to surfaces, preventing analysis, near the isoelectric point of 5.82. However, spectroscopic studies suggested the protein had an extended α-helical conformation at other pH conditions. CP30 was also shown to interact with nickel ions to form microparticles that induced elasticity in the fluid. The interactions involving these two cuticle proteins suggest new motifs that could be used in the design of tunable biomaterials. The rational design of proteins with specific covalent and non-covalent interactions with polysaccharides or ions, inspired by insect cuticle, may lead to biomaterials with enhanced mechanical properties.”
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