Enhanced water-responsive actuation of porous Bombyx mori silk.

Bombyx mori silk with a nanoscale porous architecture significantly deforms in response to changes in relative humidity. Despite the increasing amount of water adsorption and water-responsive strain with increasing porosity of the silk, there is a range of porosities that result in silk's optimal water-responsive energy density at 3.1 MJ m-3. Our findings show the possibility of controlling water-responsive materials' swelling pressure by controlling their nanoporosities.

Differential Surface Adsorption Phenomena for Conventional and Novel Surfactants Correlates with Changes in Interfacial mAb Stabilization.

Protein adsorption on surfaces can result in loss of drug product stability and efficacy during the production, storage, and administration of protein-based therapeutics. Surface-active agents (excipients) are typically added in protein formulations to prevent undesired interactions of proteins on surfaces and protein particle formation/aggregation in solution. The objective of this work is to understand the molecular-level competitive adsorption mechanism between the monoclonal antibody (mAb) and a commercially used excipient, polysorbate 80 (PS80), and a novel excipient, N-myristoyl phenylalanine-N-polyetheramine diamide (FM1000). The relative rate of adsorption of PS80 and FM1000 was studied by pendant bubble tensiometry. We find that FM1000 saturates the interface faster than PS80. Additionally, the surface-adsorbed amounts from X-ray reflectivity (XRR) measurements show that FM1000 blocks a larger percentage of interfacial area than PS80, indicating that a lower bulk FM1000 surface concentration is sufficient to prevent protein adsorption onto the air/water interface. XRR models reveal that with an increase in mAb concentration (0.5-2.5 mg/mL: IV based formulations), an increased amount of PS80 concentration (below critical micelle concentration, CMC) is required, whereas a fixed value of FM1000 concentration (above its relatively lower CMC) is sufficient to inhibit mAb adsorption, preventing mAb from co-existing with surfactants on the surface layer. With this observation, we show that the CMC of the surfactant is not the critical factor to indicate its ability to inhibit protein adsorption, especially for chemically different surfactants, PS80 and FM1000. Additionally, interface-induced aggregation studies indicate that at minimum surfactant concentration levels in protein formulations, fewer protein particles form in the presence of FM1000. Our results provide a mechanistic link between the adsorption of mAbs at the air/water interface and the aggregation induced by agitation in the presence of surfactants.

Design Strategies to Tune the Structural and Mechanical Properties of Synthetic Collagen Hydrogels.

As an important component of biomaterials, collagen provides three-dimensional scaffolds and biological cues for cell adhesion and proliferation in tissue engineering. Recombinant collagen-like proteins, which were initially discovered in Streptococcus pyogenes and produced in heterologous hosts, have been chemically and genetically engineered for biomaterial applications. However, existing collagen-like proteins do not form gels, limiting their utility as biomaterials. Here, we present a series of rationally designed collagen-like proteins composed of a trimerization domain, triple-helical domains with various lengths, and a pair of heterotrimeric coiled-coil sequences attached to the N- and C-termini as adhesive ends. These designed proteins fold into triple helices and form self-supporting gels. As the triple-helical domains are lengthened, the gels become less stiff, pore sizes increase, and structural anisotropy decreases. Moreover, cell-culture assay confirms that the designed proteins are noncytotoxic. This study provides a design strategy for collagen-based biomaterials. The sequence variations reveal a relationship between the protein primary structure and material properties, where variations in the cross-linking density and association energies define the gelation of the protein network.

Tuning water-responsiveness with Bombyx mori silk-silica nanoparticle composites.

Bombyx (B.) mori silk's water-responsive actuation correlates to its high ß-sheet crystallinity. In this research, we demonstrated that stiff silica nanoparticles can mimic the role of dispersed ß-sheet nanocrystals and dramatically increase amorphous silk's water-responsive actuation energy density to ∼700 kJ m-3.

ß-Sheet Nanocrystals Dictate Water Responsiveness of Bombyx Mori Silk.

Water-responsive (WR) materials that strongly swell and shrink in response to changes in relative humidity (RH) have shown a great potential to serve as high-energy actuators for soft robotics and new energy-harvesting systems. However, the design criteria governing the scalable and high-efficiency WR actuation remain unclear, and thus inhibit further development of WR materials for practical applications. Nature has provided excellent examples of WR materials that contain stiff nanocrystalline structures that can be crucial to understand the fundamentals of WR behavior. This work reports that regenerated Bombyx (B.) mori silk can be processed to increase ß-sheet crystallinity, which dramatically increases the WR energy density to 1.6 MJ m-3 , surpassing that of all known natural muscles, including mammalian muscles and insect muscles. Interestingly, the maximum water sorption decreases from 80.4% to 19.2% as the silk's ß-sheet crystallinity increases from 19.7% to 57.6%, but the silk's WR energy density shows an eightfold increase with higher fractions of ß-sheets. The findings of this study suggest that high crystallinity of silk reduces energy dissipation and translates the chemical potential of water-induced pressure to external loads more efficiently during the hydration/dehydration processes. Moreover, the availability of B. mori silk opens up possibilities for simple and scalable modification and production of powerful WR actuators.

Impact of Surface Amphiphilicity on the Interfacial Behavior of Janus Particle Layers under Compression.

Langmuir monolayers of silica/gold Janus particles with two different degrees of amphiphilicity have been examined to study the significance of particle surface amphiphilicity on the structure and mechanical properties of the interfacial layers. The response of the layers to the applied compression provides insight into the nature and strength of the interparticle interactions. Different collapse modes observed for the interfacial layers are linked to the amphiphilicity of Janus particles and their configuration at the interface. Molecular dynamics simulations on nanoparticles with similar contact angles provide insight on the arrangement of particles at the interface and support our conclusion that the interfacial configuration and collapse of anisotropic particles at the air/water interface are controlled by particle amphiphilicity.

Thermoresponsive Protein-Engineered Coiled-Coil Hydrogel for Sustained Small Molecule Release.

Thermoresponsive hydrogels are used for an array of biomedical applications. Lower critical solution temperature-type hydrogels have been observed in nature and extensively studied in comparison to upper critical solution temperature (UCST)-type hydrogels. Of the limited protein-based UCST-type hydrogels reported, none have been composed of a single coiled-coil domain. Here, we describe a biosynthesized homopentameric coiled-coil protein capable of demonstrating a UCST. Microscopy and structural analysis reveal that the hydrogel is stabilized by molecular entanglement of protein nanofibers, creating a porous matrix capable of binding the small hydrophobic molecule, curcumin. Curcumin binding increases the α-helical structure, fiber entanglement, mechanical integrity, and thermostability, resulting in sustained drug release at physiological temperature. This work provides the first example of a thermoresponsive hydrogel comprised of a single coiled-coil protein domain that can be used as a vehicle for sustained release and, by demonstrating UCST-type behavior, shows promise in forging a relationship between coiled-coil protein-phase behavior and that of synthetic polymer systems.

Long-Range Self-Assembly via the Mutual Lorentz Force of Plasmon Radiation.

Long-range interactions often proceed as a sequence of hopping through intermediate, statistically favored events. Here, we demonstrate predictable mechanical dynamics of particles that arise from the Lorentz force between plasmons. Even if the radiation is weak, the nonconservative Lorentz force produces stable locations perpendicular to the plasmon oscillation; over time, distinct patterns emerge. Experimentally, linearly polarized light illumination leads to the formation of 80 nm diameter Au nanoparticle chains, perpendicularly aligned, with lengths that are orders of magnitude greater than their plasmon near-field interaction. There is a critical intensity threshold and optimal concentration for observing self-assembly.

Protein Engineered Triblock Polymers Composed of Two SADs: Enhanced Mechanical Properties and Binding Abilities.

Recombinant methods have been used to engineer artificial protein triblock polymers composed of two different self-assembling domains (SADs) bearing one elastin (E) flanked by two cartilage oligomeric matrix protein coiled-coil (C) domains to generate CEC. To understand how the two C domains improve small molecule recognition and the mechanical integrity of CEC, we have constructed CL44AECL44A, which bears an impaired CL44A domain that is unstructured as a negative control. The CEC triblock polymer demonstrates increased small molecule binding and ideal elastic behavior for hydrogel formation. The negative control CL44AECL44A does not exhibit binding to small molecule and is inelastic at lower temperatures, affirming the favorable role of C domain and its helical conformation. While both CEC and CL44AECL44A assemble into micelles, CEC is more densely packed with C domains on the surface enabling the development of networks leading to hydrogel formation. Such protein engineered triblock copolymers capable of forming robust hydrogels hold tremendous promise for biomedical applications in drug delivery and tissue engineering.

'Reverse' Hofmeister effects on the sol-gel transition rates for an α-helical peptide-PEG bioconjugate.

We examine the dynamics of the sol-gel transition for end-functionalized linear- and 4-arm-peptides bioconjugated to poly-ethylene glycol (PEG) in aqueous environments with increasingly chaotropic (Cl- < Br- < I-) anions. A 23-amino acid peptide sequence is rationally designed to self-assemble upon folding into the ordered α-helical conformation due to the hydrophobic effect. We use Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy (ATR-FTIR) to quantify the ensemble average reversible secondary structure transitions as a function of electrolyte concentration and specific ion effects along the Hofmeister series. Subsequently, microrheology is used to quantify the kinetics of the gelation process, as it relates to folding and specific ion interactions. Our key findings were non-intuitive. We observe the faster evolution of the gel transitions in systems with more chaotropic anions. For our peptides in aqueous solution, "water-structuring" ions yield faster assembly behavior with a viscoelastic exponent, n, closer to unity representing self-assemblies that are Rouse-like. In contrast, ions that are "water-breaking" resulted in smaller viscoelastic exponents where self-assembly dynamics result in a viscoelastic exponent that suggests polymer entanglements.

RGD-Targeted Liposome Binding and Uptake on Breast Cancer Cells Is Dependent on Elastin Linker Secondary Structure.

The linker between the targeting moiety and the nanoparticle is often overlooked when engineering targeted drug delivery vehicles. We hypothesized that pH-triggered conformational changes of an elastin-like peptide (ELP) linker, with repeating VPGVG sequences, could alter the binding affinity of the well-established targeting moiety arginine-glycine-aspartic acid (RGD), which is known to enhance the delivery of nanoparticles to tumor cells via integrin overexpression. The pH change from blood (pH 7.4) to the tumor environment (pH 6) was used to elicit a conformational change in the ELP linker, as described by circular dichroism. Atomic force microscopy confirmed that RGD-ELP resulted in stronger adhesion to both MDA-MB-231 and HCC1806 breast cancer cells at pH 6 relative to pH 7.4. No change in adhesion force was measured as a function of pH for the non-neoplastic MCF-10A cell line and the nontargeting GDR-ELP peptide. This translated to significant binding and uptake of RGD-ELP modified liposomes at pH 6.0 relative to pH 7.4. These results indicate that the pH-triggered conformational structure of the ELP linker shifts RGD-mediated cancer cell targeting from non-active (pH 7.4) to active (pH 6). The reversible shift in ELP secondary structure may be used to engineer targeted drug delivery vehicles with tunable uptake.

Mechanical Stability of Polystyrene and Janus Particle Monolayers at the Air/Water Interface.

The compressional instability of particle-laden air/water interfaces is investigated with plain and surface-anisotropic (Janus) particles. We hypothesize that the amphiphilic nature of Janus particles leads to both anisotropic particle-particle and particle-interface interactions that can yield particle films with unique collapse mechanisms. Analysis of Langmuir isotherms and microstructural characterization of the homogeneous polystyrene particle films during compression reveal an interfacial buckling instability followed by folding, which is in good agreement with predictions from classical elasticity theory. In contrast, Janus particle films exhibit a different behavior during compression, where the collapse mode occurs through the subduction of the Janus particle film. Our results suggest that particle-laden films comprised of surface-anisotropic particles can be engineered to evolve new material properties.

Collapse of Particle-Laden Interfaces under Compression: Buckling vs Particle Expulsion.

Colloidal particles can bind to fluid interfaces with a capillary energy that is thousands of times the thermal energy. This phenomenon offers an effective route to emulsion and foam stabilization where the stability is influenced by the phase behavior of the particle-laden interface under deformation. Despite the vast interest in particle-laden interfaces, the key factors that determine the collapse of such an interface under compression have remained relatively unexplored. In this study, we illustrate the significance of the particle surface wettability and presence of electrolyte in the subphase on interparticle interactions at the interface and the resulting collapse mode. Various collapse mechanisms including buckling, particle expulsion, and multilayer formation are reported and interpreted in terms of particle-particle and particle-interface interactions.

Adsorption of rationally designed "surf-tides" to a liquid-crystal interface.

The interfacial adsorption of proteins in surfactant laden systems occurs both in nature and industrial processing, yet much of the fundamental behavior behind these systems is still not well understood. We report the development of a system that monitors optical transitions of a liquid-crystalline/aqueous interface to examine the dynamics of adsorption of two rationally designed model peptide molecules. The two molecules synthesized in this study were both designed to become surface-active upon folding and contain the same net charge of +3, but one of the peptides, K-2.5, has its three charges separated by 2.5 amino acids as compared to K-6.0, which has its three charges separated by 6 amino acids. Our study examines the roles that surfactant adsorption, peptide charge distribution and secondary structure have on the relative adsorption dynamics of these two models peptides onto a fluid/fluid interface. Using the optical detection of molecular adsorption and image analysis of these events, we obtain quantitative information about the dynamics as a function of the charge spacing and initial peptide concentration. We show that both peptides initially follow a diffusion-limited adsorption model onto the interface. Additionally, our results suggest that the K-6.0 peptides demonstrate enhanced adsorption kinetics, where the enhanced rates are a consequence of the well-folded adsorbed state and spatial distribution on the surface. These findings provide further insights into the role that charge spacing has on secondary structure and subsequently the dynamics of adsorption, while developing a versatile system capable of extracting quantitative information from a simple inexpensive optical system.

Integration of surface-active, periodically sequenced peptides into lipid-based microbubbles.

The development of microbubbles toward functional, "theranostic" particles requires the incorporation of constituents with high binding specificity and therapeutic efficacy. Integrating peptides or proteins into the shell of lipid-based microbubbles can provide a means to access both receptor-ligand interactions and therapeutic properties. Simultaneously, peptides or proteins can define the characteristic monolayer mechanics of lipid bubbles and eliminate the need for post-bubble generation modification. The ability to engineer peptide sequences de novo that effectively partition into the bubble monolayer remains parametrically daunting. This work contributes to this effort using two simple amphipathic helical peptides that examine the role of local electrostatics and secondary structure. The two periodically sequenced peptides both have three positive charges, but peptide "K-2.5" spaces those charges 2.5 amino acids apart, while peptide "K-6.0" spaces the charges six amino acids apart. Size populations were determined for bubbles containing each peptide species using light scattering, and a quantitative method was developed to clearly define the fraction of peptides binding onto the microbubble monolayer. The impact of both the initial peptide concentration and the zwitterionic:anionic lipid ratio on peptide binding was also evaluated. Our results indicate that the lipid ratio affected only K-6.0 binding, which appears to be an outcome of the greater ensemble average α-helical population of the K-6.0. These findings provide further insights into the role of charge separation on peptide secondary structure, establishing a simple design metric for peptide binding onto microbubble systems.

Structure-Dependent Water Responsiveness of Protein Block Copolymers.

Biological water-responsive (WR) materials are abundant in nature, and they are used as mechanical actuators for seed dispersal by many plants such as wheat awns and pinecones. WR biomaterials are of interest for applications as high-energy actuators, which can be useful in soft robotics or for capturing energy from natural water evaporation. Recent work on WR silk proteins has shown that ß-sheet nanocrystalline domains with high stiffness correlate with the high WR actuation energy density, but the fundamental mechanisms to drive water responsiveness in proteins remain poorly understood. Here, we design, synthesize, and study protein block copolymers consisting of two α-helical domains derived from cartilage oligomeric matrix protein coiled-coil (C) flanking an elastin-like peptide domain (E), namely, CEC. We use these protein materials to create WR actuators with energy densities that outperform mammalian muscle. To elucidate the effect of structure on WR actuation, CEC was compared to a variant, CECL44A, in which a point mutation disrupts the α-helical structure of the C domain. Surprisingly, CECL44A outperformed CEC, showing higher energy density and less susceptibility to degradation after repeated cycling. We show that CECL44A exhibits a higher degree of intermolecular interactions and is stiffer than CEC at high relative humidity (RH), allowing for less energy dissipation during water responsiveness. These results suggest that strong intermolecular interactions and the resulting, relatively steady protein structure are important for water responsiveness.

STAT6 mutations enriched at diffuse large B-cell lymphoma relapse reshape the tumor microenvironment.

Diffuse large B-cell lymphoma (DLBCL) relapses in approximately 40% of patients following frontline therapy. We reported that STAT6D419 mutations are enriched in relapsed/refractory DLBCL (rrDLBCL) samples, suggesting that JAK/STAT signaling plays a role in therapeutic resistance. We hypothesized that STAT6D419 mutations can improve DLBCL cell survival by reprogramming the microenvironment to sustain STAT6 activation. Thus, we investigated the role of STAT6D419 mutations on DLBCL cell growth and its microenvironment. We found that phospho-STAT6D419N was retained in the nucleus longer than phospho-STAT6WT following IL-4 stimulation, and STAT6D419N recognized a more restricted DNA-consensus sequence than STAT6WT. Upon IL-4 induction, STAT6D419N expression led to a higher magnitude of gene expression changes, but in a more selective list of gene targets compared with STATWT. The most significantly expressed genes induced by STAT6D419N were those implicated in survival, proliferation, migration, and chemotaxis, in particular CCL17. This chemokine, also known as TARC, attracts helper T-cells to the tumor microenvironment, especially in Hodgkin's lymphoma. To this end, in DLBCL, phospho-STAT6+ rrDLBCL cells had a greater proportion of infiltrating CD4+ T-cells than phospho-STAT6- tumors. Our findings suggest that STAT6D419 mutations in DLBCL lead to cell autonomous changes, enhanced signaling, and altered composition of the tumor microenvironment.

Tether-supported biomembranes with α-helical peptide-based anchoring constructs.

The strict requirement of constructing a native lipid environment to preserve the structure and functionality of membrane proteins is the starting constraint when building biomaterials and sensor systems from these biomolecules. To enhance the viability of supported biomembranes systems and build new ligand display interfaces, we apply rationally designed peptides partitioned into the lipid bilayer interface. Peptides designed to form membrane-spanning α-helical anchoring domains are synthesized using solid-phase peptide synthesis. K(3)A(4)L(2)A(7)L(2)A(3)K(2)-FITC is synthesized on the 100 mg scale for use as a biomembrane anchoring molecule, where orthogonal side-chain modifications allow us to introduce probes enabling peptide localization within supported bilayers. The peptides are found to form α-helical domains within liposomes as assessed with circular dichroism spectroscopy. These peptides are designed to be incorporated into lipid bilayers supported by microspheres and serve as biomembrane anchoring moieties to amino-terminated surfaces. Here, the silica bead surface (4.7 µm diameter) is activated with homobifunctional NHS-PEG(3000)-NHS as "polymer cushion" spacers. This tethering to a subset of the K(3)A(4)L(2)A(7)L(2)A(3)K(2)-FITC molecules present in the bilayer is achieved by the fusion of liposomes followed by coupling of the peptide amino groups to the NHS presented from the silica microsphere surfaces. The biomembrane distributions of tethered and untethered K(3)A(4)L(2)A(7)L(2)A(3)K(2)-FITC are probed with confocal microscopy and are found to give 3D reconstructions consistent with largely homogeneous supported biomembranes. The fluidity of the untethered fraction of peptides within supported membranes is quantified using the fluorescence recovery after photobleaching (FRAP) technique. The presence of the PEG(3000) polymer cushion facilitated a 28.9% increase in peptide diffusivity over untethered bilayers at the lowest peptide to lipid ratio we examined. We show that rationally designed peptide-based anchors can be used to tether lipid bilayers, creating a polymer-cushioned lipid microenvironment on surfaces with high lateral mobility and facilitating the development of a new platform for ligand displays.

Bionanocomposites: differential effects of cellulose nanocrystals on protein diblock copolymers.

We investigate the effects of mixing a colloidal suspension of tunicate-derived cellulose nanocrystals (t-CNCs) with aqueous colloidal suspensions of two protein diblock copolymers, EC and CE, which bear two different self-assembling domains (SADs) derived from elastin (E) and the coiled-coil region of cartilage oligomeric matrix protein (C). The resulting aqueous mixtures reveal improved mechanical integrity for the CE+t-CNC mixture, which exhibits an elastic gel network. This is in contrast to EC+t-CNC, which does not form a gel, indicating that block orientation influences the ability to interact with t-CNCs. Surface analysis and interfacial characterization indicate that the differential mechanical properties of the two samples are due to the prevalent display of the E domain by CE, which interacts more with t-CNCs leading to a stronger network with t-CNCs. On the other hand, EC, which is predominantly C-rich on its surface, does not interact as much with t-CNCs. This suggests that the surface characteristics of the protein polymers, due to folding and self-assembly, are important factors for the interactions with t-CNCs, and a significant influence on the overall mechanical properties. These results have interesting implications for the understanding of cellulose hydrophobic interactions, natural biomaterials and the development of artificially assembled bionanocomposites.

Compositional control of higher order assembly using synthetic collagen peptides.

We present the case of a two-component collagen peptide hydrogel that self-assembles through noncovalent electrostatic interactions. Natural collagen materials, such as those of connective tissue or the basement membrane, assemble in a hierarchic fashion. Similarly, the synthetic peptides presented here proceed from monomer to trimer to fiber and, finally, to a hydrogel. By varying stoichiometry and concentration, we are able to dissect the stages of higher order assembly. Insight gained from this study will improve the molecular design of biomimetic materials.