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.

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.

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.

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.

Effect of the C-terminal amino acid of the peptide on the structure and mechanical properties of alginate-peptide hydrogels across length-scales.

Alginate is a natural anionic polysaccharide that exhibits excellent biocompatibility and biodegradability. Alginate hydrogels have many different applications in the field of regenerative medicine especially when peptides are conjugated to the alginate backbone. Here, we systematically investigate the effect of six arginine-glycine-aspartic acid (RGD)-containing peptides, G6KRGDY/S, A6KRGDY/S and V6KRGDY/S, on the macroscopic and microscopic physical properties and spatial organization of alginate-peptides hydrogels. Using rheology, small angle X-ray scattering and nanoindentation measurements we show a strong correlation between the macroscopic-bulk properties and the microscopic-local properties of the alginate-peptide hydrogels. Furthermore, our results indicate that the identity of the amino acid at the C-terminal of the peptide plays a major role in determining the structure and mechanical properties of the hydrogel across length-scales, where the presence of tyrosine (Y) terminated peptides introduce more junction-zones and consequently larger stiffness than those terminated with serine (S).

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).

The dual role of MamB in magnetosome membrane assembly and magnetite biomineralization.

Magnetospirillum gryphiswaldense MSR-1 synthesizes membrane-enclosed magnetite (Fe3 O4 ) nanoparticles, magnetosomes, for magnetotaxis. Formation of these organelles involves a complex process comprising key steps which are governed by specific magnetosome-associated proteins. MamB, a cation diffusion facilitator (CDF) family member has been implicated in magnetosome-directed iron transport. However, deletion mutagenesis studies revealed that MamB is essential for the formation of magnetosome membrane vesicles, but its precise role remains elusive. In this study, we employed a multi-disciplinary approach to define the role of MamB during magnetosome formation. Using site-directed mutagenesis complemented by structural analyses, fluorescence microscopy and cryo-electron tomography, we show that MamB is most likely an active magnetosome-directed transporter serving two distinct, yet essential functions. First, MamB initiates magnetosome vesicle formation in a transport-independent process, probably by serving as a landmark protein. Second, MamB transport activity is required for magnetite nucleation. Furthermore, by determining the crystal structure of the MamB cytosolic C-terminal domain, we also provide mechanistic insight into transport regulation. Additionally, we present evidence that magnetosome vesicle growth and chain formation are independent of magnetite nucleation and magnetic interactions respectively. Together, our data provide novel insight into the role of the key bifunctional magnetosome protein MamB, and the early steps of magnetosome formation.

Short and Soft: Multidomain Organization, Tunable Dynamics, and Jamming in Suspensions of Grafted Colloidal Cylinders with a Small Aspect Ratio.

The yet virtually unexplored class of soft colloidal rods with a small aspect ratio is investigated and shown to exhibit a very rich phase and dynamic behavior, spanning from liquid to nearly melt state. Instead of the nematic order, these short and soft nanocylinders alter their organization with increasing concentration from isotropic liquid with random orientation to small domains with preferred local orientation and eventually a multidomain arrangement with a local orientational order. The latter gives rise to a kinetically suppressed state akin to structural glass with detectable terminal relaxation, which, on further increasing concentration, reveals features of hexagonally packed order as in ordered block copolymers. The respective dynamic response comprises four regimes, all above the overlapping concentration of 0.02 g/mL:(I) from 0.03 to 0.1 g/mol, the system undergoes a liquid-to-solidlike transition with a structural relaxation time that grows by 4 orders of magnitude. (II) From 0.1 to 0.2 g/mL, a dramatic slowing-down is observed and is accompanied by an evolution from isotropic to a multidomain structure. (III) Between 0.2 and 0.6 g/mol, the suspensions exhibit signatures of shell interpenetration and jamming, with the colloidal plateau modulus depending linearly on concentration. (IV) At 0.74 g/mL, in the densely jammed state, the viscoelastic signature of hexagonally packed cylinders from microphase-separated block copolymers is detected. These properties set short and soft nanocylinders apart from long colloidal rods (with a large aspect ratio) and provide insights for fundamentally understanding the physics in this intermediate soft colloidal regime and for tailoring the flow properties of nonspherical soft colloids.

Hydrogels composed of hyaluronic acid and dendritic ELPs: hierarchical structure and physical properties.

Hydrogels that mimic the native extracellular matrix were prepared from hyaluronic acid (HA) and amine-terminated dendritic elastin-like peptides (denELPs) of generations 1, 2, and 3 (G1, 2, and 3) as crosslinking units. The physical properties of the hydrogels were investigated by rheology, scanning electron microscopy, swelling tests, small-angle X-ray scattering (SAXS), and model drug loading and release assays. Hydrogel properties depended on the generation number of the denELP, which contained structural segments based on the repeating GLPGL pentamer. Hydrogels with higher generation denELPs (G2 and 3) showed similar properties, but those prepared from G1 denELPs were rheologically weaker, had a larger mesh size, absorbed less model drug, and released the drug more quickly. Interestingly, most of the HA_denELP hydrogels studied here remained transparent upon gelation, but after lyophilization and addition of water retained opaque, "solid-like" regions for up to 4 d during rehydration. This rehydration process was carefully evaluated through time-course SAXS studies, and the phenomenon was attributed to the formation of pre-coacervates in the gel-forming step, which slowly swelled in water during rehydration. These findings provide important insights into the behavior of ELP-based hydrogels, in which physical crosslinking of the ELP domains can be controlled to tune mechanical properties, highlighting the potential of HA_denELP hydrogels as biomaterials.

Self-Assembled Nanostructures Regulate H2S Release from Constitutionally Isomeric Peptides.

We report here on three constitutionally isomeric peptides, each of which contains two glutamic acid residues and two lysine residues functionalized with S-aroylthiooximes (SATOs), termed peptide-H2S donor conjugates (PHDCs). SATOs decompose in the presence of cysteine to generate hydrogen sulfide (H2S), a biological signaling gas with therapeutic potential. The PHDCs self-assemble in aqueous solution into different morphologies, two into nanoribbons of different dimensions and one into a rigid nanocoil. The rate of H2S release from the PHDCs depends on the morphology, with the nanocoil-forming PHDC exhibiting a complex release profile driven by morphological changes promoted by SATO decomposition. The nanocoil-forming PHDC mitigated the cardiotoxicity of doxorubicin more effectively than its nanoribbon-forming constitutional isomers as well as common H2S donors. This strategy opens up new avenues to develop H2S-releasing biomaterials and highlights the interplay between structure and function from the molecular level to the nanoscale.

Tuning the mechanical properties of alginate-peptide hydrogels.

Alginate, a polysaccharide that gels in the presence of divalent ions, has been used in the field of regenerative medicine to facilitate cell growth in impaired tissues by providing an artificial bio-surrounding similar to the natural extra cellular matrix (ECM). Here, we present a systematic investigation of the effect of three arginine-glycine-aspartic acid (RGD)-containing peptides, G6KRGDY, A6KRGDY and V6KRGDY, on the physical properties of alginate-peptide hydrogels. Rheology measurements showed that the storage modulus of the alginate-A6KRGDY and alginate-V6KRGDY gels is an order of magnitude higher than that of the alginate-G6KRGDY gel. Small angle X-ray scattering (SAXS) measurements suggest that the difference in the mechanical properties of the gels is due to the formation of larger peptide junction zones in addition to the ones formed by calcium ions. These findings indicate that the peptides' ability to self-assemble in aqueous solution is a significant factor in tuning the stiffness of the alginate/peptide hybrid hydrogels.

Dendritic Elastin-like Peptides: The Effect of Branching on Thermoresponsiveness.

Elastin-like peptides (ELPs) have been used widely to confer thermoresponsive characteristics onto various materials, but to this point mostly linear ELPs have been studied. A class of linear and dendritic (branched) ELPs based on the GLPGL pentamer repeat unit was synthesized using an on-resin divergent strategy. The effect of peptide topology on the transition temperature (Tt) was examined using circular dichroism to study the peptide secondary structure transition and turbidity to measure the macroscopic phase transition (coacervation). Secondary structure transitions showed no dependence on topology, but a higher Tt was observed for dendritic peptides than for linear peptides with the same number of GLPGL repeats. The data support a phase transition model that consists of two neighboring processes: a secondary structure transition, related to intramolecular interactions, followed by coacervation, associated with intermolecular interactions.

Molecular design for growth of supramolecular membranes with hierarchical structure.

Membranes with hierarchical structure exist in biological systems, and bio-inspired building blocks have been used to grow synthetic analogues in the laboratory through self-assembly. The formation of these synthetic membranes is initiated at the interface of two aqueous solutions, one containing cationic peptide amphiphiles (PA) and the other containing the anionic biopolymer hyaluronic acid (HA). The membrane growth process starts within milliseconds of interface formation and continues over much longer timescales to generate robust membranes with supramolecular PA-HA nanofibers oriented orthogonal to the interface. Computer simulation indicates that formation of these hierarchically structured membranes requires strong interactions between molecular components at early time points in order to generate a diffusion barrier between both solutions. Experimental studies using structurally designed PAs confirm simulation results by showing that only PAs with high ζ potential are able to yield hierarchically structured membranes. Furthermore, the chemical structure of such PAs must incorporate residues that form ß-sheets, which facilitates self-assembly of long nanofibers. In contrast, PAs that form low aspect ratio nanostructures interact weakly with HA and yield membranes that exhibit non-fibrous fingering protrusions. Furthermore, experimental results show that increasing HA molecular weight decreases the growth rate of orthogonal nanofibers. This result is supported by simulation results suggesting that the thickness of the interfacial contact layer generated immediately after initiation of self-assembly increases with polymer molecular weight.

Electrostatic control of structure in self-assembled membranes.

Self-assembling peptide amphiphiles (PAs) can form hierarchically ordered membranes when brought in contact with aqueous polyelectrolytes of the opposite charge by rapidly creating a diffusion barrier composed of filamentous nanostructures parallel to the plane of the incipient membrane. Following this event, osmotic forces and charge complexation template nanofiber growth perpendicular to the plane of the membrane in a dynamic self-assembly process. In this work, we show that this hierarchical structure requires massive interfacial aggregation of PA molecules, suggesting the importance of rapid diffusion barrier formation. Strong PA aggregation is induced here through the use of heparin-binding PAs with heparin and also with polyelectrolytes of varying charge density. Small angle X-ray scattering shows that in the case of weak PA-polyelectrolyte interaction, membranes formed display a cubic phase ordering on the nanoscale that likely results from clusters of PA nanostructures surrounded by polyelectrolyte chains.

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.

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.

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.

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.