Biomacromolecules

Peptide-brush polymers prepared via graft-through polymerization of peptide-based olefinic monomers form Protein-Like Polymers (PLPs) with demonstrated utility as chemical biology tools for modulating intracellular protein--protein interactions and are in development as a emerging class of therapeutic. This favorable bioactivity has been achieved in part by mimicking some of the structural and functional features of proteins, such as their dynamic, globular solution-phase structures, ability to insert into lipid membranes and enter cells, and tendency to enforce -helicity in peptide side chains. However, because structure leads to function, insight into the structure of these proteomimetic polymers, particularly their overall tertiary structure and the extent to which this influences the brush peptide structure, is incomplete. To further this understanding, we prepared a set of brush polymers incorporating the trpzip2 peptide sequence, commonly used as a model system for protein folding. We examine these polymers via circular dichroism spectroscopy, infrared spectroscopy, and small-angle X-ray scattering, coupled with insights from molecular dynamics simulations. Here, a loss of the {beta}-hairpin structure of the monomer is observed regardless of polymer composition, and simulations corroborate that the polymer backbone destabilizes local secondary structure, restricting peptide motion and altering the accessible free-energy landscape. This study provides insight into design rules governing these synthetic polymer-based proteomimetics.

Hydrogels are cross-linked polymeric networks with wide applications in drug delivery, tissue engineering, biosensing, and environmental remediation. These hydrogels additionally host living cells, small molecules and biological propagules, which further expand the applications of these materials. However, most if not all fabrication methods require covalent modifications. In this work, for the first time, we demonstrate that polymer mixtures can access an additional material state beyond the conventionally described homogeneous and two-phase regimes. By deliberately selecting polymers with a known propensity to phase separate and formulating compositions far from the binodal boundary, the system transitions directly into a mechanically stable hydrogel. We demonstrate this technique using a model system of poly (ethylene glycol) (PEG) and dextran (DEX). We have systematically characterized the hydrogels through FTIR, MALDI-TOF to discern the molecular compositions of the hydrogels. We also modulate the optical transparency of these hydrogels by varying the molecular weight of the polymers. These experimental findings are supplemented with coarse grained (CG) simulation insights to investigate the mechanistic origins of phase separation propensity with varying molecular weights of dextran. We utilized coexisting densities in the two phases using CG simulations to predict the role of dextran molecular weight on the partitioning of PEG and DEX in the two phases. Finally, we exploit the fabricated hydrogels ability to encapsulate live cells, antibiotics and plant seeds. We anticipate that this ATPS-based fabrication technique will provides a scalable, crosslinker-free route to multifunctional hydrogels enabling advanced applications in drug delivery and responsive materials.

Ternary composite systems formed by lactoferrin (LF), sodium alginate (Alg), and Fe(II) were designed to investigate their potential as an iron delivery platform with enhanced protein stability. The ternary LF-Alg-Fe (LAF) composites demonstrated distinct structures depending on the LF to Alg ratio and the Fe(II) concentrations. At an LF to Alg ratio of 8:2 and final Fe concentrations between 20-30 mM, the system formed complexes stabilized by electrostatic interactions. Whereas Alg-rich formulations formed hydrogels stabilized by Alg-Fe(II) egg-box cross-linking. Rheological analysis and swelling behavior indicated a higher mechanical strength in LF-rich complexes and stronger network integrity in Alg-rich hydrogels, while intermediate LF/Alg ratios showed weaker structures overall. Fourier-transform infrared spectroscopy (FTIR) spectra showed no changes in functional groups or polymer structures after composite formation, confirming composite formation via non-covalent interactions. Thermal studies indicated that these ternary systems improved LF stability, evidenced by preserved secondary structure after heating using circular dichroism (CD), and an increased denaturation temperature compared with free LF in differential scanning calorimetry (DSC). In addition, in LF-rich formulations the Fe(II) release in aqueous solution was [~]50% while in Alg-rich formulations it was much lower (< 10%). LF-Alg-Fe composites exhibit distinct structures governed by protein-polysaccharide interactions and iron-mediated cross-linking, providing a potential strategy for protein stabilization and iron fortification in food systems.

Physically assembled, supramolecular hydrogels often are formed by weak bonds, producing useful viscoelastic properties that are reminiscent of soft tissues within the human body yet presenting challenges in stability under physiological conditions. In this work, we establish an approach for creating self-assembling, supramolecular hydrogels based on a peptide-polymer conjugate (PPC) as a three-dimensional (3D) cell culture platform. The viscoelastic properties of these hydrogels, including thermoresponsiveness and shear-thinning, were measured using rheometry. Building on these observations, we then developed a method for the encapsulation and culture of human wound healing cells, fibroblasts, within the PPC hydrogels, observing both excellent cell viability and material stability over time. The modulation of stiffness by temperature variation and the reversible nature of these hydrogels imparts unique properties for both cell encapsulation and harvesting and offers potential for a variety of biomedical applications, from 3D cell culture systems to therapeutic delivery.

Chemical zymogens of three different types were established herein around protein cysteinome, in each case converting the protein thiol into a disulfide linkage: zero length Z0, polyethylene glycol based ZPEG, and ZLA that features a fast-depolymerizing fuse polymer. The latter was a polydisulfide based on a naturally occurring water-soluble lipoic acid. Three zymogen designs were applied to cysteinyl proteases and a kinase and in each case, enzymatic activity was successfully masked in full and reactivated by small molecule reducing agents. However, only ZLA could be reactivated by protein activators, demonstrating that the macromolecular fuse escapes the steric bulk created by the protein globule, collects activation signal in solution, and relays it to the enzyme active site. This afforded first-in-class chemical zymogens that are activated via protein-protein interactions. For ZLA, we also document a "chain transfer" bioconjugation mechanism and a unique zymogen exchange reaction between two proteins.

Pluronic (Plu) hydrogels containing Pluronic diacrylate (PluDA) have become popular matrices to encapsulate bacteria in engineered living materials. For this purpose, 30 wt% Plu/PluDA hydrogels with variable fraction of covalently crosslinkable PluDA in the hydrogel composition are used. The degree of covalent crosslinking and the consequent different mechanical properties of the hydrogels have been shown to affect bacteria growth, but a systematic investigation of the mechanical response of the hydrogels is still missing. Here we study the rheological response of 30 wt.% Plu/PluDA hydrogels with increasing PluDA fraction between 0 and 1. We quantify the range of viscoelastic properties that can be covered in this system by varying in the PluDA fraction. We present stress relaxation and creep-recovery experiments, and analyze the variation of the critical yield strain/stress, relaxation and recovery parameters of Plu/PluDA hydrogels as function of the covalent crosslinking degree using the Burgers and Weilbull models. We expect this study to help users of Plu/PluDA hydrogels to estimate the mechanical properties of their systems, and eventually to correlate them with the behaviour of bacteria in future Plu/PluDA devices of similar composition.

This article synthesized and characterized a novel hydrogel, which is formed using maleimide-thiol conjugation. Two precursors chitosan functionalized thiol groups and dextran functionalized maleimide groups prepared and characterized by NMR. The hydrogel studied by gelation time, swelling studies, viscoelastic properties, degradation rate. From gelation time result, we found that formed hydrogel gelation time could be changed with diffident weight percentage of precursors. Based on references, we found the best formular for the gelation and it was also determined for other studies. The swelling study indicated hydrogel has good flexibility and the degradation test indicated hydrogel is biodegradable. The viscoelastic test indicated hydrogel is elastic solid. From these studies, this a novel hydrogel could be potential for biomedical applications.

Protein-based microparticles are promising materials for applications such as biocatalysis and biomolecular capture, yet their fabrication by existing techniques remains challenging due to protein denaturation or lack of spatial control. Here, we present a method for synthesizing microscale protein-based materials by chemically crosslinking biomolecular condensates. Leveraging the liquid-liquid phase separation behavior of intrinsically disordered RGG domains, we sequestered RGG-tagged fusion proteins into droplets, then we solidified them into porous microparticles using the homobifunctional, amine-reactive crosslinker BS3. By modulating protein concentration and condensate coalescence, we controlled microparticle size from <1 to >40 {micro}m. We then demonstrated three encodable functionalities: We used the SpyCatcher/SpyTag system to capture cargo proteins, we crosslinked core-shell condensates to generate microparticles with controlled spatial organization, and we immobilized a thermostable alcohol dehydrogenase with 31% retained enzymatic activity. These POMPOMS (protein-based, self-organized microparticles of multifunctional significance) represent a sustainable, tunable platform for versatile protein-based materials.

Hydrogels have a wide range of applications such as in biomedicine, cosmetics and soft electronics. Compared to polymer hydrogels based on covalent bonding, protein hydrogels offer distinct advantages owing to their biocompatibility and better access to molecular engineering. However, pure and natural protein hydrogels have been seldom reported except for structural proteins like collagen and silk fibrin. Here, we report the unusual ability and mechanism of a unique natural enzyme, lipoate-protein ligase A (LplA) of E. coli to self-assemble into a stimuli-responsive and reversible hydrogel of the low critical solution temperature (LCST) type. This is the first globular and catalytic protein found to form a hydrogel in response to temperature, pH and the presence of ions. Protein structure based analysis reveals the key residues responsible for the gel formation and mutational studies confirms the essential roles of hydrogen bonding between the C-terminal domains and electrostatic interactions in the N-terminal domains. Characterization of phase transitions of wild type LplA and its mutants using small angle X-ray scattering (SAXS) yields details of the gelation process from initial dimer formation over a pre-gel-state to full network development. Further electron microscopic analyses and modeling of SAXS data suggest an unusual interlinked ladder-like structure of the macroscopic crosslinking network with dimers as ladder steps. The unique features of this first reported protein hydrogel may open up hitherto inaccessible applications, especially those taking advantage of the inherent catalytic activity of LplA.

Polymer modification of antibodies has attracted attention for enhancing antibody functionality. Modification with polyethylene glycol (PEG) or biopolymers such as antibody-oligonucleotide conjugates has been investigated. Although direct effects at the modification site have been understood, distal effects of modifications on intrinsic antibody functions such as antigen-binding and Fc receptor-binding activities remain poorly characterized. In this study, we prepared site-specifically PEGylated trastuzumab to evaluate the effects of the modification site and the size of PEG chains on antibody functions. Reporter gene assays revealed a significant reduction in antibody-dependent cell cytotoxicity (ADCC) activity in a site- and size-dependent manner. Both the reduction in interaction with the antigen and Fc{gamma}RIIIa were observed in a size-dependent manner. Interestingly, the effect of the modification site was only observed for ADCC activity, which suggests that the higher-order structure of the modified antibody plays a critical role in effector function. These results provide molecular design guidelines for the polymer modification of antibodies.

Dynamic covalent hydrogels formed through reversible acylhydrazone crosslinking have emerged as promising injectable biomaterials. However, a fundamental gap remains in understanding how the macromolecular structure of oxidized polysaccharides (OxPs) governs gelation kinetics and how these kinetics pathways translate into material properties and cellular responses. We address this question by developing an acylhydrazone hydrogel library composed of alginate adipohydrazide crosslinked with oxidized alginate (OxA) or oxidized dextran (OxD), two reactive aldehyde-bearing polymers with comparable chemical functionality but fundamentally distinct backbone structure. By varying polysaccharide type, oxidation degree, and reaction pH, we decoupled the effects of chemical functionality from macromolecular structure and established quantitative structure-kinetics-property-function relationships. OxD-based hydrogels undergo rapid, largely pH-independent gelation, whereas OxA-based systems display pronounced pH-dependent kinetics with significantly delayed network formation under physiological pH. These differences in gelation kinetics and OxPs macromolecular structures lead to marked variations in hydrogel mechanics, including stiffness, stress relaxation, stability, injectability, and post-injection recovery. Importantly, differences in gelation kinetics modulate cell-matrix interactions in three-dimensional culture. Slowly forming OxA hydrogels maintained rounded chondrocyte shape, while rapidly gelling OxD networks induced transient cell elongation. Mesenchymal stem cells displayed similar shapes regardless of gelation kinetics, indicating cell-type-specific responses to matrix formation dynamics.

Load-bearing tissues, such as muscle and cartilage, exhibit mechanical properties that often combine high elasticity, high toughness and fast recovery, despite their different stiffness ([~]100 kPa for muscles and one to several MPa for cartilage).1-7 The advance in protein engineering and protein mechanics has made it possible to engineer protein-based biomaterials to mimic soft load-bearing tissues, such as muscles.8-10 However, it is challenging to engineer protein biomaterials to achieve the mechanical properties exhibited by stiff tissues, such as articular cartilage,6,11 or to develop stiff synthetic extracellular matrices for cartilage stem/progenitor cell differentiation12. By employing physical entanglements13 of protein chains and force-induced protein unfolding,14,15 here we report the engineering of a highly tough and stiff protein hydrogel to mimic articular cartilage. By crosslinking an engineered artificial elastomeric protein from its unfolded state, we introduced chain entanglement into the hydrogel network. Upon renaturation, the entangled protein chain network and forced protein unfolding entailed this single network protein hydrogel with superb mechanical properties in both tensile and compression tests, showing a Youngs modulus of [~]0.7 MPa and toughness of 250 kJ/m3 in tensile testing; and [~]1.7 MPa in compressive modulus and toughness of 3.2 MJ/m3. The energy dissipation in both tensile and compression tests is reversible and the hydrogel can recovery its mechanical properties rapidly. Moreover, this hydrogel can withstand a compression stress of >60 MPa without failure, amongst the highest compressive strength achieved by a hydrogel. These properties are comparable to those of articular cartilage, making this protein hydrogel a novel cartilage-mimetic biomaterial. Our study opened up a new potential avenue towards engineering protein hydrogel-based substitute for articular cartilage, and may also help develop protein biomaterials with superb mechanical properties for applications in soft actuators and robotics.

Withdrawal StatementThis manuscript has been withdrawn following a formal review by the George Mason Universitys Office of Research Integrity and Assurance.

Hydrogels are widely used as three-dimensional cell culture systems to understand the impact of cellular mechanotransduction for tissue engineering applications. Photoinitiated thiol-ene click chemistry is a commonly utilized hydrogel crosslinking mechanism that provides spatial and temporal control over hydrogel network formation and resulting mesh size and compressive properties. Despite historically documented efficiency as step-growth reactions, these reactions do not always proceed as predicted. To understand the impact of cell confinement and microenvironmental mechanics on cellular function, thiol-ene network formation must be thoroughly characterized. To this end, the objective of this work was to investigate the crosslinking dynamics to determine hydrogel network formation as assessed via mesh size and mechanical properties using a pentenoate-functionalized hyaluronic acid thiol-ene reaction. Hydrogel parameters including polymer concentration and thiol:-ene crosslinker molar ratio were modulated (4, 6, or 8 polymer weight percent and 0.15:1, 0.5:1, or 1:1 molar ratio of thiol groups to reactive -ene groups) to tune network properties including shear storage modulus and relative mesh size. Molecular Dynamics (MD) simulations were used to simulate the thiol-ene crosslinking reaction and establish a method for predicting thiol-ene reaction efficiency. Lastly, the feasibility of this hydrogel system for in vitro modeling was confirmed via assessment of metabolic activity of encapsulated primary human meniscal cells.

Polyampholyte gel is a perfect physics model to mimic condensed state of proteins. We have studied the hierarchical dynamics of polyampholyte gels by dynamic light scattering. In addition to the normal gel mode, which indicates the gel elasticity, we also discovered a new mode with a stretched exponential decay with the stretched exponent {beta} = 1/3, and a diffusive exponential decay, which indicates the coupled motion between counterion and the polyampholyte backbone. After dialysis to low salt concentration, the coupled motion of the counterion will go away, so that there are only two modes. Combined with a newly developed theory, we attribute this stretched exponential mode to hierarchical dynamics of the segments between two crosslinking junctions, whose segmental distribution obeys Poisson distribution. As salt concentration inside the gel increases, {beta} decreases from 0.38 to 0.33, which is consistent with theoretical results. The gel with the molar charge ratio R=1, which is at the charge balance point, has the highest value {beta} = 0.38. As long as R deviates further away from the charge balance point from either side, the {beta} values decrease. When the gel is 100% positive charged, their dynamic light scattering results will go back to that of the normal polyelectrolyte gels.

Thiol-ene click chemistry is a powerful tool for designing hydrogels mimicking the mechanical and biochemical properties of 3D cellular microenvironments. The high selectivity of thiol-norbornene step-growth polymerization enables precise control of crosslinking mechanism, circumventing the alkene homopolymerization present in other systems that can prevent encapsulated cell spreading. Limited stress relaxation of a dynamically-crosslinked norbornene-modified hyaluronic acid (NorHA) hydrogel employing a thiol-norbornene photoclick reaction led us to investigate the prevalence of norbornene homopolymerization in this supposed click reaction. Norbornene conversion was quantified in multiple thiol plus norbornene-modified polymer system permutations, revealing higher norbornene conversion than expected for 1:1 thiol-ene addition. We showed that decreasing the number of norbornenes per NorHA chain (f) mitigated network formation via norbornene homopolymerization. Dynamic hydrogels fabricated with NorHA of f = 8 (Nor8HA) exhibited 93.0 {+/-} 1.6% relaxation, while those fabricated with NorHA of f = 40 (Nor40HA) achieved only 42.3 {+/-} 0.1% relaxation. As early as day 3 of culture, Nor8HA hydrogels facilitated spreading of encapsulated human mesenchymal stromal cells (hMSCs) into a spindle-like morphology (aspect ratio: 2.95 {+/-} 0.38), while Nor40HA hydrogels appeared to constrain cells into a spherical or compact star morphology (aspect ratio: 1.22 {+/-} 0.01). Inference of a single-cell morphological space derived from a shape-matching distance metric validated the two distinct hMSC morphological phenotypes primarily associated with polymer f. Despite its widespread use as a click reaction, radical-mediated thiol-norbornene crosslinking was found to not be stoichiometric in dilute aqueous conditions used to fabricate hydrogels. Altering network topology through polymer f enabled the rescue of hydrogel dynamic behavior and encapsulated hMSC spreading, despite the presence of norbornene homopolymerization, highlighting the need to consider network-level properties when designing engineered cellular microenvironments.

Supramolecular architecture formation by the self-assembly of proteins and nucleic acids is well studied. Yet, the spontaneous organization of oligosaccharides, the most common polymers in nature, is less explored. Here, using inulin as a model, we identify the "sweet spot" length for oligosaccharide assembly. Inulin forms discrete spheres in a concentration-dependent manner. Size-based fractionation displayed markedly different aggregation morphologies. Based on these findings, we believe that carbohydrates could become an important source for novel self-assembling materials.

Withdrawal StatementThis manuscript has been withdrawn following a formal review by the George Mason Universitys Office of Research Integrity and Assurance.

The remarkable elastic properties of polymers are ultimately due to their molecular structure, but the relation between the macroscopic and molecular properties is often difficult to establish, in particular for (bio)polymers that contain hydrogen bonds, which can easily rearrange upon mechanical deformation. Here we show that two-dimensional infrared spectroscopy on polymer films in a miniature stress tester sheds new light on how the hydrogen-bond structure of a polymer is related to its visco-elastic response. We study thermoplastic polyurethane, a block copolymer consisting of hard segments of hydrogen-bonded urethane groups embedded in a soft matrix of polyether chains. The conventional infrared spectrum shows that upon deformation, the number of hydrogen bonds increases, a process that is largely reversible. However, the 2DIR spectrum reveals that the distribution hydrogen-bond strengths becomes slightly narrower after a deformation cycle, due to the disruption of weak hydrogen bonds, an effect that could explain the strain-cycle induced softening (Mullins effect) of polyurethane. These results show how rheo-2DIR spectroscopy can bridge the gap between the molecular structure and the macroscopic elastic properties of (bio)polymers.

Interest in utilizing amyloids to develop biomaterials is increasing due to their potential for biocompatibility, unique assembling morphology, mechanical stability, and biophysical properties. However, challenges include the complexity of peptide chemistry and the practical techniques required for processing amyloids into bulk materials. In this work, two decapeptides with fibrillar and globular morphologies were selected, blended with poly(ethylene oxide), and fabricated into composite mats via electrospinning. Notable enhancements in mechanical properties were observed, attributed to the uniform distribution of the decapeptide assemblies within the PEO matrix. Morphological differences, such as the production of thinner nanofibers, are attributed to the increased conductivity from the zwitterionic nature of the decapeptides. Blend rheology and post-processing analysis revealed how processing might affect the amyloid aggregation and secondary structure of the peptides. Both decapeptides demonstrated good biocompatibility and strong antioxidant activity, indicating their potential for safe and effective use as biomaterials. By evaluating these interdependencies, this research lays the foundation for understanding the structure-property-processing relationships of peptide-polymer blends and highlights the strong potential for developing applications in biotechnology.