Decoupling Charge and Side Chain Effects in Hierarchical Organization of Cationic PFX Peptide and Alginate.

We have successfully created self-assembled membranes by combining positively charged (Pro-X-(Phe-X)5-Pro) PFX peptides with negatively charged alginate. These PFX/alginate membranes were formed by three different peptides that contain either X = Arginine (R), Histidine (H), or Ornithine (O) as their charged amino acid. The assemblies were compared to membranes that were previously reported by us composed of X = lysine (K). This study enabled us to elucidate the impact of amino acids' specific interactions on membrane formation. SEM, SAXS, and cryo-TEM measurements show that although K, R, H, and O may have a similar net charge, the specific traits of the charged amino acid is an essential factor in determining the hierarchical structure of alginate/PFX self-assembled membranes.

Glucose-Triggered Gelation of Supramolecular Peptide Nanocoils with Glucose-Binding Motifs.

Peptide self-assembly is a powerful tool to prepare functional materials at the nanoscale. Often, the resulting materials have high aspect-ratio, with intermolecular ß-sheet formation underlying 1D fibrillar structures. Inspired by dynamic structures in nature, peptide self-assembly is increasingly moving toward stimuli-responsive designs wherein assembled structures are formed, altered, or dissipated in response to a specific cue. Here, a peptide bearing a prosthetic glucose-binding phenylboronic acid (PBA) is demonstrated to self-assemble into an uncommon nanocoil morphology. These nanocoils arise from antiparallel ß-sheets, with molecules aligned parallel to the long axis of the coil. The binding of glucose to the PBA motif stabilizes and elongates the nanocoil, driving entanglement and gelation at physiological glucose levels. The glucose-dependent gelation of these materials is then explored for the encapsulation and release of a therapeutic agent, glucagon, that corrects low blood glucose levels. Accordingly, the release of glucagon from the nanocoil hydrogels is inversely related to glucose level. When evaluated in a mouse model of severe acute hypoglycemia, glucagon delivered from glucose-stabilized nanocoil hydrogels demonstrates increased protection compared to delivery of the agent alone or within a control nanocoil hydrogel that is not stabilized by glucose.

Potent inhibition of MMP-9 by a novel sustained-release platform attenuates left ventricular remodeling following myocardial infarction.

Sustained drug-release systems prolong the retention of therapeutic drugs within target tissues to alleviate the need for repeated drug administration. Two major caveats of the current systems are that the release rate and the timing cannot be predicted or fine-tuned because they rely on uncontrolled environmental conditions and that the system must be redesigned for each drug and treatment regime because the drug is bound via interactions that are specific to its structure and composition. We present a controlled and universal sustained drug-release system, which comprises minute spherical particles in which a therapeutic protein is affinity-bound to alginate sulfate (AlgS) through one or more short heparin-binding peptide (HBP) sequence repeats. Employing post-myocardial infarction (MI) heart remodeling as a case study, we show that the release of C9-a matrix metalloproteinase-9 (MMP-9) inhibitor protein that we easily bound to AlgS by adding one, two, or three HBP repeats to its sequence-can be directly controlled by modifying the number of HBP repeats. In an in vivo study, we directly injected AlgS particles, which were bound to C9 through three HBP repeats, into the left ventricular myocardium of mice following MI. We found that the particles substantially reduced post-MI remodeling, attesting to the sustained, local release of the drug within the tissue. As the number of HBP repeats controls the rate of drug release from the AlgS particles, and since C9 can be easily replaced with almost any protein, our tunable sustained-release system can readily accommodate a wide range of protein-based treatments.

Enantioselective disruption of cellulose nanocrystal self-assembly into chiral nematic phases in D-alanine solutions.

The chiral environment of enantiomerically pure D-alanine solutions is observed to disrupt and modify the entropy-driven assembly of cellulose nanocrystals (CNCs) into a chiral nematic mesophase. The effect is specific to D-alanine and cannot be attributed to the adsorption of alanine molecules (neither D- nor L-alanine) onto the CNC particles.

Self-assembly at the interface of λ-carrageenan and amphiphilic and cationic peptides: More than meets the eye.

Self-assembly of macroscopic membranes at the interface between self-assembling peptides and aqueous polymer solutions of opposite charge has been explored mostly due to the membranes' unique hierarchical structure of three distinct regions, including a layer of perpendicular fibers. We report here on the formation and characterization of self-assembled membranes made with λ-carrageenan and the cationic ß-sheet peptides, Pro-Lys-(Phe-Lys)5-Pro (PFK). Using SAXS, SEM, ITC, and rheology, we compared these membranes' morphology and physical properties to membranes made with alginate. We recognized that the polysaccharide's single chain conformation, its solution's viscosity, the potential of hydrogen bonding and electrostatic interactions between the polysaccharides and the peptides charged groups, and the strength of these interactions all affect the properties of the resulting membranes. As a result, we identified that an interplay between the polymer-peptide strength of interactions and the stiffness of the polysaccharide's single chain could be used as a route to control the structure-function relationship of the membranes. These results provide valuable information for creating guidelines to design self-assembly membranes with specific properties.

APPI-Derived Cyclic Peptide Enhances Aß42 Aggregation and Reduces Aß42-Mediated Membrane Destabilization and Cytotoxicity.

An amyloid precursor protein inhibitor (APPI) and amyloid beta 42 (Aß42) are both subdomains of the human transmembrane amyloid precursor protein (APP). In the brains of patients with Alzheimer's disease (AD), Aß42 oligomerizes into aggregates of various sizes, with intermediate, low-molecular-weight Aß42 oligomers currently being held to be the species responsible for the most neurotoxic effects associated with the disease. Strategies to ameliorate the toxicity of these intermediate Aß42 oligomeric species include the use of short, Aß42-interacting peptides that either inhibit the formation of the Aß42 oligomeric species or promote their conversion to high-molecular-weight aggregates. We therefore designed such an Aß42-interacting peptide that is based on the ß-hairpin amino acid sequence of the APPI, which exhibits high similarity to the ß-sheet-like aggregation site of Aß42. Upon tight binding of this 20-mer cyclic peptide to Aß42 (in a 1:1 molar ratio), the formation of Aß42 aggregates was enhanced, and consequently, Aß42-mediated cell toxicity was ameliorated. We showed that in the presence of the cyclic peptide, interactions of Aß42 with both plasma and mitochondrial membranes and with phospholipid vesicles that mimic these membranes were inhibited. Specifically, the cyclic peptide inhibited Aß42-mediated mitochondrial membrane depolarization and reduced Aß42-mediated apoptosis and cell death. We suggest that the cyclic peptide modulates Aß42 aggregation by enhancing the formation of large aggregates─as opposed to low-molecular-weight intermediates─and as such has the potential for further development as an AD therapeutic.

Activating hidden signals by mimicking cryptic sites in a synthetic extracellular matrix.

Cryptic sites are short signaling peptides buried within the native extracellular matrix (ECM). Enzymatic cleavage of an ECM protein reveals these hidden peptide sequences, which interact with surface receptors to control cell behavior. Materials that mimic this dynamic interplay between cells and their surroundings via cryptic sites could enable application of this endogenous signaling phenomenon in synthetic ECM hydrogels. We demonstrate that depsipeptides ("switch peptides") can undergo enzyme-triggered changes in their primary sequence, with proof-of-principle studies showing how trypsin-triggered primary sequence rearrangement forms the bioadhesive pentapeptide YIGSR. We then engineered cryptic site-mimetic synthetic ECM hydrogels that experienced a cell-initiated gain of bioactivity. Responding to the endothelial cell surface enzyme aminopeptidase N, the inert matrix transformed into an adhesive synthetic ECM capable of supporting endothelial cell growth. This modular system enables dynamic reciprocity in synthetic ECMs, reproducing the natural symbiosis between cells and their matrix through inclusion of tunable hidden signals.

Charge Regulation of Poly(acrylic acid) in Solutions of Non-Charged Polymer and Colloids.

Weak polyelectrolytes (WPEs) are responsive materials used as active charge regulators in a variety of applications, including controlled release and drug delivery in crowded bio-related and synthetic environments. In these environments, high concentrations of solvated molecules, nanostructures, and molecular assemblies are ubiquitous. Here, we investigated the effect of high concentrations of non-adsorbing, short chains of poly(vinyl alcohol), PVA, and colloids dispersed by the very same polymers on charge regulation (CR) of poly(acrylic acid), PAA. PVA does not interact with PAA (throughout the full pH range) and thus can be used to examine the role of non-specific (entropic) interactions in polymer-rich environments. Titration experiments of PAA (mainly 100 kDa in dilute solutions, no added salt) were carried out in high concentrations of PVA (13-23 kDa, 5-15 wt%) and dispersions of carbon black (CB) decorated by the same PVA (CB-PVA, 0.2-1 wt%). The calculated equilibrium constant (and pKa) was up-shifted in PVA solutions by up to ~0.9 units and down-shifted in CB-PVA dispersions by ~0.4 units. Thus, while solvated PVA chains increase the charging of the PAA chains, as compared to PAA in water, CB-PVA particles reduce PAA charging. To investigate the origins of the effect, we analyzed the mixtures using small-angle X-ray scattering (SAXS) and cryo-TEM imaging. The scattering experiments revealed re-organization of the PAA chains in the presence of the solvated PVA but not in the CB-PVA dispersions. These observations clearly indicate that the acid-base equilibrium and the degree of ionization of PAA in crowded liquid environments is affected by the concentration, size, and geometry of seemingly non-interacting additives, probably due to depletion and excluded volume interactions. Thus, entropic effects that do not depend on specific interactions should be taken into consideration when designing functional materials in complex fluid environments.

Native Glucagon Amyloids Catalyze Key Metabolic Reactions.

Glucagon is a prominent peptide hormone, playing central roles in the regulation of glucose blood-level and lipid metabolism. Formation of glucagon amyloid fibrils has been previously reported, although no biological functions of such fibrils are known. Here, we demonstrate that glucagon amyloid fibrils catalyze biologically important reactions, including esterolysis, lipid hydrolysis, and dephosphorylation. In particular, we found that glucagon fibrils catalyze dephosphorylation of adenosine triphosphate (ATP), a core metabolic reaction in cell biology. Comparative analysis of several glucagon variants allowed mapping the catalytic activity to an enzymatic pocket-like triad formed at the glucagon fibril surface, comprising the histidyl-serine domain at the N-terminus of the peptide. This study may point to previously unknown physiological roles and pathological consequences of glucagon fibrillation and supports the hypothesis that catalytic activities of native amyloid fibrils play functional roles in human physiology and disease.

Effects of Non-Ionic Micelles on the Acid-Base Equilibria of a Weak Polyelectrolyte.

Weak polyelectrolytes (WPEs) are widely used as pH-responsive materials, pH modulators and charge regulators in biomedical and technological applications that involve multi-component fluid environments. In these complex fluids, coupling between (often weak) interactions induced by micelles, nanoparticles and molecular aggregates modify the pKa as compared to that measured in single component solutions. Here we investigated the effect of coupling between hydrogen bonding and excluded volume interactions on the titration curves and pKa of polyacrylic acid (PAA) in solutions comprising PEO-based micelles (Pluronics and Brij-S20) of different size and volume fraction. Titration experiments of dilute, salt-free solutions of PAA (5 kDa, 30 kDa and 100 kDa) at low degree of polymer ionization (α < 0.25) drive spatial re-organization of the system, reduce the degree of ionization and consequentially increase the pKa by up to ~0.7 units. These findings indicate that the actual degree of ionization of WPEs measured in complex fluids is significantly lower (at a given pH) than that measured in single-component solutions.

Surfactant-Mediated Co-Existence of Single-Walled Carbon Nanotube Networks and Cellulose Nanocrystal Mesophases.

Hybrids comprising cellulose nanocrystals (CNCs) and percolated networks of single-walled carbon nanotubes (SWNTs) may serve for the casting of hybrid materials with improved optical, mechanical, electrical, and thermal properties. However, CNC-dispersed SWNTs are depleted from the chiral nematic (N*) phase and enrich the isotropic phase. Herein, we report that SWNTs dispersed by non-ionic surfactant or triblock copolymers are incorporated within the surfactant-mediated CNC mesophases. Small-angle X-ray measurements indicate that the nanostructure of the hybrid phases is only slightly modified by the presence of the surfactants, and the chiral nature of the N* phase is preserved. Cryo-TEM and Raman spectroscopy show that SWNTs networks with typical mesh size from hundreds of nanometers to microns are distributed equally between the two phases. We suggest that the adsorption of the surfactants or polymers mediates the interfacial interaction between the CNCs and SWNTs, enhancing the formation of co-existing meso-structures in the hybrid phases.

Avidity observed between a bivalent inhibitor and an enzyme monomer with a single active site.

Although myriad protein-protein interactions in nature use polyvalent binding, in which multiple ligands on one entity bind to multiple receptors on another, to date an affinity advantage of polyvalent binding has been demonstrated experimentally only in cases where the target receptor molecules are clustered prior to complex formation. Here, we demonstrate cooperativity in binding affinity (i.e., avidity) for a protein complex in which an engineered dimer of the amyloid precursor protein inhibitor (APPI), possessing two fully functional inhibitory loops, interacts with mesotrypsin, a soluble monomeric protein that does not self-associate or cluster spontaneously. We found that each inhibitory loop of the purified APPI homodimer was over three-fold more potent than the corresponding loop in the monovalent APPI inhibitor. This observation is consistent with a suggested mechanism whereby the two APPI loops in the homodimer simultaneously and reversibly bind two corresponding mesotrypsin monomers to mediate mesotrypsin dimerization. We propose a simple model for such dimerization that quantitatively explains the observed cooperativity in binding affinity. Binding cooperativity in this system reveals that the valency of ligands may affect avidity in protein-protein interactions including those of targets that are not surface-anchored and do not self-associate spontaneously. In this scenario, avidity may be explained by the enhanced concentration of ligand binding sites in proximity to the monomeric target, which may favor rebinding of the multiple ligand binding sites with the receptor molecules upon dissociation of the protein complex.

Hyaluronan (HA)-inspired glycopolymers as molecular tools for studying HA functions.

Hyaluronic acid (HA), the only non-sulphated glycosaminoglycan, serves numerous structural and biological functions in the human body, from providing viscoelasticity in tissues to creating hydrated environments for cell migration and proliferation. HA is also involved in the regulation of morphogenesis, inflammation and tumorigenesis through interactions with specific HA-binding proteins. Whilst the physicochemical and biological properties of HA have been widely studied for decades, the exact mechanisms by which HA exerts its multiple functions are not completely understood. Glycopolymers offer a simple and precise synthetic platform for the preparation of glycan analogues, being an alternative to the demanding synthetic chemical glycosylation. A library of homo, statistical and alternating HA glycopolymers were synthesised by reversible addition-fragmentation chain transfer polymerisation and post-modification utilising copper alkyne-azide cycloaddition to graft orthogonal pendant HA monosaccharides (N-acetyl glucosamine: GlcNAc and glucuronic acid: GlcA) onto the polymer. Using surface plasmon resonance, the binding of the glycopolymers to known HA-binding peptides and proteins (CD44, hyaluronidase) was assessed and compared to carbohydrate-binding proteins (lectins). These studies revealed potential structure-binding relationships between HA monosaccharides and HA receptors and novel HA binders, such as Dectin-1 and DEC-205 lectins. The inhibitory effect of HA glycopolymers on hyaluronidase (HAase) activity was also investigated suggesting GlcNAc- and GlcA-based glycopolymers as potential HAase inhibitors.

Control over size, shape, and photonics of self-assembled organic nanocrystals.

The facile fabrication of free-floating organic nanocrystals (ONCs) was achieved via the kinetically controlled self-assembly of simple perylene diimide building blocks in aqueous medium. The ONCs have a thin rectangular shape, with an aspect ratio that is controlled by the content of the organic cosolvent (THF). The nanocrystals were characterized in solution by cryogenic transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering. The ONCs retain their structure upon drying, as was evidenced by TEM and atom force microscopy. Photophysical studies, including femtosecond transient absorption spectroscopy, revealed a distinct influence of the ONC morphology on their photonic properties (excitation energy transfer was observed only in the high-aspect ONCs). Convenient control over the structure and function of organic nanocrystals can enhance their utility in new and developed technologies.

Crescent-Shaped Supramolecular Tetrapeptide Nanostructures.

Self-assembly of amphiphilic peptide-based building blocks gives rise to a plethora of interesting nanostructures such as ribbons, fibers, and tubes. However, it remains a great challenge to employ peptide self-assembly to directly produce nanostructures with lower symmetry than these highly symmetric motifs. We report here our discovery that persistent and regular crescent nanostructures with a diameter of 28 ± 3 nm formed from a series of tetrapeptides with the general structure AdKSKSEX (Ad = adamantyl group, KS = lysine residue functionalized with an S-aroylthiooxime (SATO) group, E = glutamic acid residue, and X = variable amino acid residue). In the presence of cysteine, the biological signaling gas hydrogen sulfide (H2S) was released from the SATO units of the crescent nanostructures, termed peptide-H2S donor conjugates (PHDCs), reducing levels of reactive oxygen species (ROS) in macrophage cells. Additional in vitro studies showed that the crescent nanostructures alleviated cytotoxicity induced by phorbol 12-myristate-13-acetate more effectively than common H2S donors and a PHDC of a similar chemical structure, AdKSKSE, that formed short nanoworms instead of nanocrescents. Cell internalization studies indicated that nanocrescent-forming PHDCs were more effective in reducing ROS levels in macrophages because they entered into and remained in cells better than nanoworms, highlighting how nanostructure morphology can affect bioactivity in drug delivery.

A combined experimental and computational approach reveals how aromatic peptide amphiphiles self-assemble to form ion-conducting nanohelices.

Reported here is a combined experimental-computational strategy to determine structure-property-function relationships in persistent nanohelices formed by a set of aromatic peptide amphiphile (APA) tetramers with the general structure K S XEK S , where KS= S-aroylthiooxime modified lysine, X = glutamic acid or citrulline, and E = glutamic acid. In low phosphate buffer concentrations, the APAs self-assembled into flat nanoribbons, but in high phosphate buffer concentrations they formed nanohelices with regular twisting pitches ranging from 9-31 nm. Coarse-grained molecular dynamics simulations mimicking low and high salt concentrations matched experimental observations, and analysis of simulations revealed that increasing strength of hydrophobic interactions under high salt conditions compared with low salt conditions drove intramolecular collapse of the APAs, leading to nanohelix formation. Analysis of the radial distribution functions in the final self-assembled structures led to several insights. For example, comparing distances between water beads and beads representing hydrolysable KS units in the APAs indicated that the KS units in the nanohelices should undergo hydrolysis faster than those in the nanoribbons; experimental results verified this hypothesis. Simulation results also suggested that these nanohelices might display high ionic conductivity due to closer packing of carboxylate beads in the nanohelices than in the nanoribbons. Experimental results showed no conductivity increase over baseline buffer values for unassembled APAs, a slight increase (0.4 × 102 µS/cm) for self-assembled APAs under low salt conditions in their nanoribbon form, and a dramatic increase (8.6 × 102 µS/cm) under high salt conditions in their nanohelix form. Remarkably, under the same salt conditions, these self-assembled nanohelices conducted ions 5-10-fold more efficiently than several charged polymers, including alginate and DNA. These results highlight how experiments and simulations can be combined to provide insight into how molecular design affects self-assembly pathways; additionally, this work highlights how this approach can lead to discovery of unexpected properties of self-assembled nanostructures.

Time matters for macroscopic membranes formed by alginate and cationic ß-sheet peptides.

Hierarchically ordered planar and spherical membranes (sacs) were constructed using amphiphilic and cationic ß-sheet peptides that spontaneously assembled together with negatively charged alginate solution. The system was found to form either a fully developed membrane structure with three distinct regions including characteristic perpendicular fibers or a non-fully developed contact layer lacking these standing fibers, depending on the peptide age, membrane geometry and membrane incubation time. The morphological differences were found to strongly depend on fairly-long incubation time frames that influenced both the peptide's intrinsic alignment and the reaction-diffusion process taking place at the interface. A three-stage mechanism was suggested and key parameters affecting the development process were identified. Stability tests in biologically relevant buffers confirmed the suitability of these membranes for bio applications.

Nano-to-meso structure of cellulose nanocrystal phases in ethylene-glycol-water mixtures.

The self-assembly and phase behavior of cellulose nanocrystals (CNCs) in binary liquid mixtures of ethylene-glycol (EG):water was investigated. Our findings indicate that a small fraction of water delays the onset of colloidal jammed states previously reported in water-free organic solvents. Here the full phase diagram of CNCs evolves, including the chiral nematic phase (N*), characterized by long-range orientational order and non-isotropic macroscopic properties. Furthermore, the effect of the solvent-mixture composition on the properties of the CNC mesophases is found to be scale-dependent: the micron-size pitch of the N* phase decreases as the dielectric constant (εr) of the solvent mixture is reduced (higher EG content). Yet the nanometric inter-particle spacing of the CNC rods (measured using SAXS and cryo-TEM) is almost independent on the EG content. Also, unlike theoretical predictions, the transition to the biphasic regime is not sensitive to εr of the solvent mixtures and takes place at a higher CNC volume fraction than in aqueous suspensions. These observations may be rationalized by hypothesizing that vicinal water, adsorbed at the CNC surface, prevents kinetic arrest, and dictates the local dielectric constant and thus the effective diameter of the rods (via the Debye length), while εr of the liquid-mixture dominates the pitch length (micron scale) and the optical properties. These findings indicate that the water content of EG:water mixtures may be used for engineering colloidal inks where delayed kinetic arrest and jamming of the CNCs enable printing and casting of tunable, optically-active thin films and coatings.

Sensing Exposure Time to Oxygen by Applying a Percolation-Induced Principle.

The determination of food freshness along manufacturer-to-consumer transportation lines is a challenging problem that calls for cheap, simple, reliable, and nontoxic sensors inside food packaging. We present a novel approach for oxygen sensing in which the exposure time to oxygen-rather than the oxygen concentration per se-is monitored. We developed a nontoxic hybrid composite-based sensor consisting of graphite powder (conductive filler), clay (viscosity control filler) and linseed oil (the matrix). Upon exposure to oxygen, the insulating linseed oil is oxidized, leading to polymerization and shrinkage of the matrix and hence to an increase in the concentration of the electrically conductive graphite powder up to percolation, which serves as an indicator of food spoilage. In the developed sensor, the exposure time to oxygen (days to weeks) is obtained by measuring the electrical conductivity though the sensor. The sensor functionality could be tuned by changing the oil viscosity, the aspect ratio of the conductive filler, and/or the concentration of the clay, thereby adapting the sensor to monitoring the quality of food products with different sensitivities to oxygen exposure time (e.g., fish vs grain).

Effect of Crosslinker Topology on Enzymatic Degradation of Hydrogels.

Despite the widespread use of hydrogels in biomedical applications, little is known regarding the effect of crosslinker topology on hydrogel degradation. Dendritic and linear elastin-like peptides (ELPs) were used as crosslinkers for hyaluronic acid (HA) hydrogels, and their enzymatic degradation was studied using trypsin. Rheological studies revealed that hydrogels crosslinked with ELP dendrimers (HA_denELPs) degraded more slowly than those crosslinked with the otherwise equivalent linear ELPs (i.e., both molecules have the same number of pentamers and peripheral lysine residues). The origin of this phenomenon was evaluated using solution studies in which various dendritic and linear ELPs were treated with trypsin. Apart from the expected steric hindrances due to the dendritic topology, we identified the dual directionality of the peptide sequences (generated by a central branching lysine residue) and the likelihood of cleaving a productive crosslinking point as two additional contributors to the lesser degradability of HA_denELPs. Overall, these results highlight how the molecular design of crosslinker topology represents a novel strategy to tune the degradation rate of hydrogels.