Controlling topological entanglement in engineered protein hydrogels with a variety of thiol coupling chemistries

Topological entanglements between polymer chains are achieved in associating protein hydrogels through the synthesis of high molecular weight proteins via chain extension using a variety of thiol coupling chemistries, including disulfide formation, thiol-maleimide, thiol-bromomaleimide and thiol-ene. Coupling of cysteines via disulfide formation results in the most pronounced entanglement effect in hydrogels, while other chemistries provide versatile means of changing the extent of entanglement, achieving faster chain extension, and providing a facile method of controlling the network hierarchy and incorporating stimuli responsivities. The addition of trifunctional coupling agents causes incomplete crosslinking and introduces branching architecture to the protein molecules. The high-frequency plateau modulus and the entanglement plateau modulus can be tuned by changing the ratio of difunctional chain extender to the trifunctional branching unit. Therefore, these chain extension reactions show promise in delicately controlling the relaxation and mechanical properties of engineered protein hydrogels in ways that complement their design through genetic engineering

Site-specific conjugation of RAFT polymers to proteins via expressed protein ligation

Site-specific protein conjugates with RAFT polymers were synthesized using expressed protein ligation. Stable micelles were formed from both linear block copolymer and Y-shaped conjugates.DTRA (Project BA12PHM159

Effect of polymer chemistry on globular protein–polymer block copolymer self-assembly

Bioconjugates of the model red fluorescent protein mCherry and synthetic polymer blocks with different hydrogen bonding functionalities show that the chemistry of the polymer block has a large effect on both ordering transitions and the type of nanostructures formed during bioconjugate self-assembly. The phase behaviours of mCherry-b-poly(hydroxypropyl acrylate) (PHPA) and mCherry-b-poly(oligoethylene glycol acrylate) (POEGA) in concentrated aqueous solution show that changes in polymer chemistry result in increase in the order–disorder transition concentrations (C[subscript ODT]s) by approximately 10–15 wt% compared to a previously studied globular protein–polymer block copolymer, mCherry-b-poly(N-isopropylacrylamide) (PNIPAM). The C[subscript ODT]s are always minimized for symmetric bioconjugates, consistent with the importance of protein–polymer interactions in self-assembly. Both mCherry-b-PHPA and mCherry-b-POEGA also form phases that have not previously been observed in other globular protein–polymer conjugates: mCherry-b-PHPA forms a cubic phase that can be indexed to Ia[bar over 3]d and mCherry-b-POEGA displays coexistence of lamellae and a cubic Ia[bar over 3]d structure over a narrow range of concentration and temperature. Several common behaviours are also revealed by comparison of different polymer blocks. With increasing concentration and temperature, ordered phases always appear in the order lamellar, cubic/PL, and hexagonal, although not all phases are observed in all materials. High concentration solutions (near 80 wt%) also undergo a re-entrant order–disorder transition to form nematic liquid crystalline phases, regardless of the polymer block chemistry.United States. Air Force Office of Scientific Research (Award FA9550-12-0259)United States. Dept. of Energy. Office of Basic Energy Sciences (Award DE-SC0007106

Oxidatively Responsive Chain Extension to Entangle Engineered Protein Hydrogels

Engineering artificial protein hydrogels for medical applications requires precise control over their mechanical properties, including stiffness, toughness, extensibility, and stability in the physiological environment. Here we demonstrate topological entanglement as an effective strategy to robustly increase the mechanical tunability of a transient hydrogel network based on coiled-coil interactions. Chain extension and entanglement are achieved by coupling the cysteine residues near the N- and C-termini, and the resulting chain distribution is found to agree with the Jacobson–Stockmayer theory. By exploiting the reversible nature of the disulfide bonds, the entanglement effect can be switched on and off by redox stimuli. With the presence of entanglements, hydrogels exhibit a 7.2-fold enhanced creep resistance and a suppressed erosion rate by a factor of 5.8, making the gels more mechanically stable in a physiologically relevant open system. While hardly affecting material stiffness (only resulting in a 1.5-fold increase in the plateau modulus), the entanglements remarkably lead to hydrogels with a toughness of 65 000 J m^(–3) and extensibility to approximately 3000% engineering strain, which enables the preparation of tough yet soft tissue simulants. This improvement in mechanical properties resembles that from double-network hydrogels but is achieved with the use of a single associating network and topological entanglement. Therefore, redox-triggered chain entanglement offers an effective approach for constructing mechanically enhanced and responsive injectable hydrogels

Protective effect of buganjianyao decoction against IL-1βinduced degeneration of endplate chondrocytes in rats via NF-κB signaling pathway

Purpose: To investigate the protective effect of buganjianyao decoction (BJD) against IL-1β-induced degeneration of endplate chondrocytes in a rat model, and the underlying mechanism of action. Methods: Rat endplate chondrocytes were cultured in 6-well tissue culture plates. Two types of serum were used: normal serum and BJD-containing serum. The endplate chondrocytes were grouped as follows: blank group given 0.5 % FBS, induced group treated with 0.5 % volume fraction of IL-1β (10 μg/L), normal serum groups treated with 5, 10 and 20 % volume fractions of normal serum, and BJD serum groups treated with 5, 10 and 20 % volume fractions of BJD-containing serum. Cell Counting Kit8 (CCK-8) assay was used to measure the proliferation of endplate chondrocytes. The expressions of aggrecan and matrix metalloproteinase-3(MMP-3) were determined by ELISA, while reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to assay the mRNA expressions of IκB kinaseα (IKKα) and NF-κB p65. Western blotting was used measure the protein expressions of IKKα and NF-κB p65. Results: Compared with normal serum group treated with a similar volume fraction, the proliferation capacity and aggrecan expression of BJD serum group increased at 24 h and 48 h post-treatment, while expressions of MMP-3, IKKα and NF-κB p65 decreased. The effects were more pronounced in the 20 % volume fraction of BJD serum group than in the other groups. Conclusion: BJD exerts protective effect against IL-1β-induced degeneration of endplate chondrocytes via inhibition of the NF-κB signaling pathway. This finding provides an experimental basis for the potential development of BJD for the treatment of DDD

Epistatic Roles for Pseudomonas aeruginosa MutS and DinB (DNA Pol IV) in Coping with Reactive Oxygen Species-Induced DNA Damage

Pseudomonas aeruginosa is especially adept at colonizing the airways of individuals afflicted with the autosomal recessive disease cystic fibrosis (CF). CF patients suffer from chronic airway inflammation, which contributes to lung deterioration. Once established in the airways, P. aeruginosa continuously adapts to the changing environment, in part through acquisition of beneficial mutations via a process termed pathoadaptation. MutS and DinB are proposed to play opposing roles in P. aeruginosa pathoadaptation: MutS acts in replication-coupled mismatch repair, which acts to limit spontaneous mutations; in contrast, DinB (DNA polymerase IV) catalyzes error-prone bypass of DNA lesions, contributing to mutations. As part of an ongoing effort to understand mechanisms underlying P. aeruginosa pathoadaptation, we characterized hydrogen peroxide (H2O2)-induced phenotypes of isogenic P. aeruginosa strains bearing different combinations of mutS and dinB alleles. Our results demonstrate an unexpected epistatic relationship between mutS and dinB with respect to H2O2-induced cell killing involving error-prone repair and/or tolerance of oxidized DNA lesions. In striking contrast to these error-prone roles, both MutS and DinB played largely accurate roles in coping with DNA lesions induced by ultraviolet light, mitomycin C, or 4-nitroquinilone 1-oxide. Models discussing roles for MutS and DinB functionality in DNA damage-induced mutagenesis, particularly during CF airway colonization and subsequent P. aeruginosa pathoadaptation are discussed

Dynamics and mechanics of associating polymer networks

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2016.Cataloged from PDF version of thesis.Includes bibliographical references.Associating polymers have attracted much interest in a variety of applications such as selfhealing materials, biomaterials, rheological modifiers, and actuators. The interplay of polymer topology and sticker chemistry presents significant challenges in understanding the physics of associating polymers across a wide range of time and length scales. This thesis aims to provide new insights into the structure-dynamics-mechanics relationships of associating polymer networks. This thesis first examines diffusion of various types of associating polymers in the gel state through a combination of experiment and theory. By using forced Rayleigh scattering (FRS), phenomenological super-diffusion is revealed as a general feature in associating networks. Experimental findings are quantitatively explained by a simple two-state model that accounts for the interplay of chain diffusion and the dynamic association-dissociation equilibrium of polymer chains with surrounding network. Furthermore, hindered self-diffusion is shown to directly correlate with a deviation from the Maxwellian behavior in materials rheological response on the long time scale. To further understand how sticker dynamics affects the network mechanical properties, a new method referred to as "sticker diffusion and dissociation spectrometry" is developed to quantify the dissociation rate of stickers in the network junctions. It is demonstrated that sticker dissociation is a prerequisite step for sticker exchange that leads to macroscopic stress relaxation. Finally, this thesis explores the use of fluorescence recovery after photobleaching (FRAP) to measure self-diffusion of associating polymers, and a mathematical framework is established. The second part of this thesis focuses on the development of new methods of controlling the mechanical properties of associating networks through engineering the molecular structure of polymer chains. Specifically, topological entanglement is introduced into the network through extending the polymer chains to reach beyond their entanglement threshold. This strategy drastically enhances material's toughness, extensibility, creep resistance and stability in solutions. Various types of coupling chemistries are then explored to fine tune the extent of entanglement. The entanglement effect and the long-time relaxation of materials can be further controlled by introducing branching points into the macromolecules.by Shengchang Tang.Ph. D

Relaxation Processes in Supramolecular Metallogels Based on Histidine–Nickel Coordination Bonds

Understanding the quantitative relationship between the dynamic mechanical properties of associating polymer networks and the dynamics of sticker bonds represents an important problem in polymer science because materials mechanics is affected by not only the sticker bond chemistry but also the sticker position, the polymer structure, and the physical environment of the associating polymers such as concentration and solvent quality. In this study, associating networks formed by structurally well-defined linear poly­(<i>N</i>,<i>N</i>-dimethyl­acrylamide) polymers with histidine side groups in complexation with Ni²⁺ ions are chosen as a model system. “Sticker diffusion and dissociation spectrometry” is developed as a new method to quantify the dissociation dynamics of stickers within the network environment where the stickers are covalently attached to polymers above their overlap concentration. The estimated time constants for junction dissociation in gels are shown to be substantially different than the ones measured by metal exchange experiments on small-molecule junctions in the dilute solution limit. Additionally, the in-gel dissociation time constants exhibit the same temperature dependence as the network relaxation times inferred from rheological characterization, which serves as the basis for time–temperature superposition, provided that the network relaxation is governed by the dissociation kinetics of stickers. Furthermore, self-diffusion of these associating polymers is probed by forced Rayleigh scattering, and pure Fickian diffusive behavior is revealed. The characteristic time constants for all the explored dynamic processes are finally viewed in the superimposed frequency sweep spectrum, demonstrating the inherent hierarchical relaxation in associating polymer networks even with only a single type of junction functionality

Molecular anisotropy and rearrangement as mechanisms of toughness and extensibility in entangled physical gels

Dynamic networks formed by physically crosslinked, entangled polymers have emerged as self-healing, stretchable, and functional materials. Entangled associative gels with remarkable toughness and extensibility have been produced by several distinct chemical approaches, suggesting that these enhanced mechanical properties result from molecular-scale topology. Previously, artificially engineered associative proteins were designed to provide an ideal model system to investigate the role of entanglement on gel mechanics via well-defined entangled or unentangled physical gels. Herein, uniaxial strain-induced structural changes in these model gels were observed using in situ small-angle x-ray scattering (SAXS) and in situ polarized optical microscopy (POM) up to 2000% engineering strain. Anisotropic optical responses to uniaxial strain at the nano-, micro-, and macroscales suggest that stress dissipation mechanisms enable high extensibility and toughness. Nano- and microscopic anisotropy observed by SAXS indicate stretching and alignment of flexible polymer strands along the straining axis, and nonmonotonic macroscopic anisotropy observed by POM suggests relaxation within the hydrogel due to rearrangement of associative network junctions. Unentangled hydrogels exhibit low toughness and a strain-rate-dependent transition from ductile to brittle tensile behavior, which is typical for associative polymer solutions. These findings indicate that topological entanglements and the freedom of individual chains to align at the nanoscale due to junction relaxation are both critical to achieving high toughness and elongation in entangled physical gels.National Science Foundation (Grant DMR-1709315)Army Research Office (Contract W911NF-07-D-0004

Anomalous Self-Diffusion and Sticky Rouse Dynamics in Associative Protein Hydrogels

Natural and synthetic materials based on associating polymers possess diverse mechanical behavior, transport properties and responsiveness to external stimuli. Although much is known about their dynamics on the molecular and macroscopic level, knowledge of self-diffusive dynamics of the network-forming constituents remains limited. Using forced Rayleigh scattering, anomalous self-diffusion is observed in model associating protein hydrogels originating from the interconversion between species that diffuse in both the molecular and associated state. The diffusion can be quantitatively modeled using a two-state model for polymers in the gel, where diffusivity in the associated state is critical to the super diffusive behavior. The dissociation time from bulk rheology measurements was 2–3 orders of magnitude smaller than the one measured by diffusion, because the former characterizes submolecular dissociation dynamics, whereas the latter depicts single protein molecules completely disengaging from the network. Rheological data also show a sticky Rouse-like relaxation at long times due to collective relaxation of large groups of proteins, suggesting mobility of associated molecules. This study experimentally demonstrates a hierarchy of relaxation processes in associating polymer networks, and it is anticipated that the results can be generalized to other associative systems to better understand the relationship of dynamics among sticky bonds, single molecules, and the entire network