The self-assembly of amphiphilic bottlebrush block copolymers (BCPs), featuring backbones densely grafted with two types of side chains, is less well understood compared to linear BCPs. In particular, the solution self-assembly of tapered bottlebrush BCPs-cone-shaped BCPs with hydrophilic or hydrophobic tips-remains unexplored. This study investigates eight tapered and four cylindrical bottlebrush BCPs with varied ratios of hydrophobic polystyrene (PS) and hydrophilic poly(acrylic acid) (PAA) side chains, synthesized via sequential addition of macromonomers using ring-opening metathesis polymerization (SAM-ROMP). Self-assembled nanostructures formed in water were analyzed using cryogenic transmission electron microscopy, small-angle neutron scattering, and dynamic light scattering. Most BCPs generated multiple nanostructures with surface protrusions, including spherical micelles, cylindrical micelles, and vesicles, alongside transitional forms like ellipsoids and semi-vesicles. Coarse-grained molecular dynamics simulations supported the experimental findings, which revealed two distinct self-assembly pathways. The first involved micelle fusion, producing elliptical and cylindrical aggregates, sometimes forming Y-junctions. The second pathway featured micelle maturation into semivesicles, which developed into vesicles or large compound vesicles. This work provides the first experimental evidence of vesicle formation via semivesicles in bottlebrush BCPs and demonstrates the significant influence of cone directionality on self-assembly behavior in these cone-shaped polymeric amphiphiles.

Entropy-driven assembly of nematic liquid-crystal phases of cellulose nanocrystals (SCNCs) in aqueous suspensions results in the emergence of a cholesteric liquid crystalline phase (N* phase). We report that a solvated, non-adsorbing, highly branched natural polysaccharide, Gum Arabic (GA), strongly affects the assembly of the SCNCs and modifies the phase diagram: GA leads to significant crowding of the SCNC rods and induces a new liquid-liquid phase transition, where SCNC-rich and GA-rich droplets coexist. The solvated GA does not induce coagulation or gelation of the suspended SCNCs (at low concentrations of 1-3 wt% of GA). In the SCNC-rich droplets, finite-sized nematic nano-islands assemble and further evolve into cholesteric tactoids and nucleate the formation of the N* phase at significantly lower concentration (about 1.5 wt%) than in GA-free suspensions. We observe that the inter-particle distance and the chiral pitch of the N* phase are determined by the concentration of GA (for a given SCNC concentration). The resulting mesophases are characterized via transmission electron microscopy at cryogenic temperatures (cryo-TEM), small-angle X-ray scattering (SAXS), and polarized optical microscopy (POM). Our findings indicate that GA can be used to tune the phase diagram and optical properties of SCNC suspensions, and overcome kinetic barriers that lead to gelation or kinetic arrest.

We present pH-responsive colloidal paste based on brushes of poly(acrylic acid) (PAA) grafted on silica microparticles (∼4 μm) that actively modulates bulk fluid flow in packed bed columns of the colloid paste. pH-dependent charge and conformational changes of the PAA brushes in aqueous 10 mM NaNO3 solutions (pH 2-10) give an 8-fold change in flow rate (0.04-0.32 mL/min) through dynamic particle rearrangement in the paste, offering potential for flow rate modulation by pH. The colloid paste is a onsive "colloidal flow gate" that actively changes its permeability based on the aqueous solution acidity. We characterize the colloidal paste permeability by measuring pH variations of the flow rate through the paste.

Self-assembled peptide-H2S donor conjugates (PHDCs) can deliver hydrogen sulfide in vitro and in vivo, yet the link between the supramolecular nanostructure morphology and cellular uptake remains unclear. Herein, we designed constitutionally isomeric PHDCs that self-assembled in aqueous solution into either nanoribbons, nanofibers, or nanobelts with various dimensions based on cryogenic transmission electron microscopy and small-angle X-ray scattering. Nile-red loaded PHDCs showed morphology-dependent uptake by H9C2 cells based on fluorescence microscopy combined with flow cytometry and confocal imaging, where narrow, helically twisted nanoribbons entered most efficiently. All PHDCs released H2S at similar rates, but the amount of H2S released inside the cells depended on the internalization ability of each PHDC. Consistent with these results, the narrow twisted nanoribbons afforded the greatest protection against H2O2-induced oxidative stress. Overall, this study highlights how subtle molecular-level changes can influence nanostructure formation in supramolecular assemblies and ultimately affect their cellular uptake and biological activities.

Self-assembly of amphiphilic bottlebrush block copolymers (BCPs), which have backbones with two types of densely grafted side chains, is less well understood compared to their linear counterparts. Specifically, the solution self-assembly of tapered bottlebrush BCPs, which are approximately cone-shaped with specific cone directionality (hydrophilic or hydrophobic tips), remains unexplored. This work describes a series of 8 tapered and 4 cylindrical bottlebrush BCPs with varying ratios of hydrophobic polystyrene (PS) and hydrophilic poly(acrylic acid) (PAA) side chains, synthesized by the sequential addition of macromonomers ring-opening metathesis polymerization (SAM-ROMP) method. The nanostructures formed by these BCPs in water were evaluated using cryogenic transmission electron microscopy, small-angle neutron scattering, and dynamic light scattering. Results showed that most BCPs formed multiple types of nanostructures, all with surface protrusions, including spherical micelles, cylindrical micelles, and vesicles, with transitional structures between each, such as ellipsoids and semi-vesicles. Coarse-grained molecular dynamics simulations aided interpretation of the experimental data. Collectively, results revealed two distinct self-assembly pathways through which the BCPs evolved from micelles into complex nanostructures. One pathway involved micelle fusion, resulting in elliptical and cylindrical aggregates that in some cases fused further to form Y-junctions. The second pathway entailed micelle maturation into semivesicles, which subsequently formed vesicles and, at times, large compound vesicles. This study provides the first experimental evidence supporting vesicle formation via semivesicles in bottlebrush BCPs. Collectively, these findings highlight how structural parameters such as cone directionality influence self-assembly in these large, cone-shaped polymeric amphiphiles.

The stability of colloidal and nanoparticle (NPs) dispersions is a prerequisite for their utilization in advanced technologies. Yet, disposal of large volumes of NPs-rich fluid is challenging for waste management and would benefit from the ability to induce controlled aggregation and sedimentation of the NPs. Here, we demonstrate that sterically stabilized dispersions of carbonaceous NPs (Carbon Black and Single-Walled Carbon Nanotubes) in aqueous solutions of Arabinogalactan (AG) are pH responsive: the dispersions are stable at pH = 7 but coagulate at pH = 2 and separate into NPs-poor fluid and NPs-rich sediment. The sediment can be easily re-dispersed at pH = 7. The molecular and colloidal origins of the unexpected pH responsiveness of the sterically stabilized AG dispersions are investigated using surface tension measurements and Small-Angle X-ray Scattering experiments. While the pH responsiveness, along with the re-disposability, opens new pathways for reducing the ecological footprint of the NPs dispersions, the molecular origins of the observed behavior are not resolved and would require further investigation.

Coassembly of peptide biomaterials offers a compelling avenue to broaden the spectrum of hierarchically ordered supramolecular nanoscale structures that may be relevant for biomedical and biotechnological applications. In this study we present a comprehensive exploration of binary coassembly leveraging amphiphilic and oppositely charged, anionic and cationic, -sheet peptides, which may give rise to a diverse range of coassembled forms. Mixtures of the peptides exhibit a notably diminished critical assembly concentration (CAC), in comparison to the corresponding values of the pure peptides. Intriguingly, the sweet spot for coassembled fibril formation was found to require excess of the cationic peptide whereas equimolar mixtures of the peptides exhibited the maximum folding into β-sheet structures. Mixtures of the peptides coassembled sequentially from solutions at concentrations surpassing each peptide's intrinsic CAC, were also found to require a higher portion of the cationic peptide to stabilize hydrogels. This study illuminates a systematic exploration of complementary charged -sheet peptides. The results may be relevant to the fundamental understanding of such intricate assembly systems and to the formulation of peptide-based nanostructures with diverse functionalities.

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.

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.

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.

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.

Peptide self-assembly is a powerful tool to prepare functional materials at the nanoscale. Often, the resulting materials have high aspect-ratio, with intermolecular β-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 β-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.

An amyloid precursor protein inhibitor (APPI) and amyloid beta 42 (Aβ42) are both subdomains of the human transmembrane amyloid precursor protein (APP). In the brains of patients with Alzheimer's disease (AD), Aβ42 oligomerizes into aggregates of various sizes, with intermediate, low-molecular-weight Aβ42 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 Aβ42 oligomeric species include the use of short, Aβ42-interacting peptides that either inhibit the formation of the Aβ42 oligomeric species or promote their conversion to high-molecular-weight aggregates. We therefore designed such an Aβ42-interacting peptide that is based on the β-hairpin amino acid sequence of the APPI, which exhibits high similarity to the β-sheet-like aggregation site of Aβ42. Upon tight binding of this 20-mer cyclic peptide to Aβ42 (in a 1:1 molar ratio), the formation of Aβ42 aggregates was enhanced, and consequently, Aβ42-mediated cell toxicity was ameliorated. We showed that in the presence of the cyclic peptide, interactions of Aβ42 with both plasma and mitochondrial membranes and with phospholipid vesicles that mimic these membranes were inhibited. Specifically, the cyclic peptide inhibited Aβ42-mediated mitochondrial membrane depolarization and reduced Aβ42-mediated apoptosis and cell death. We suggest that the cyclic peptide modulates Aβ42 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.

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 & lambda;-carrageenan and the cationic & beta;-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.

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.

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.

Over the years, several models of triacylglycerol (TAG) molecular conformations and bulk arrangements in isotropic liquid state have been proposed and are still up for debate. Such organization has a major impact on nucleation and crystal formation. The current research aims to further explore this debate by implementing experimental methods, such as X-ray diffraction and small-angle X-ray scattering coupled with a computational method, such as molecular dynamic simulation. These techniques were used to study tristearin and triolein as models for saturated and unsaturated TAGs, respectively. Four different conformations were suggested for the two TAGs, and the results showed conformation abundancy in the order: trident (Tr) > chair (Ch) > propeller (Pr) > tuning-fork (Tf). The existence of clusters was demonstrated for both TAGs, each of which exhibited a heterogeneous distribution of conformations. The preferability to find a specific pair of conformations next to each other was analyzed and, surprisingly, it was found that Tf will preferably pair only with Tr although Tf is the preferable conformation in most crystal polymorphs. High general conversion rates from any conformation to another, and high specific conversion rates from and to the Tf conformation were calculated. It is proposed that the high conversion rates observed enable the crystallization process, despite the low proportion of Tf molecules, which are in the suitable conformation for the crystal state. The conversion rates in triolein were lower compared with tristearin. This observation was ascribed to the lower simulated temperatures and the higher rigidity of the fatty acid chain induced by the double bond. Overall, the results confirm the formation of specific structures in liquid state, which combine all previously suggested models and further expand the knowledge using experimental and computational tools.(c) 2022 Elsevier B.V. All rights reserved.

Biocompatible and biodegradable molecules capable of spontaneous self-assembly into hierarchical structures have the potential to serve as building blocks for complex scaffolds for cell culture. These scaffolds are meant to mimic the natural environment required for successful cell growth. Thus, they must be precisely designed and present specific mechanical and structural properties along with required biological and chemical cues. Combining the hierarchical structure and chemical functionality of a peptide with the stability of a polymer has led to the formation of complex and stable biomaterials. Among these hybrid biomaterials, macroscopic membranes self-assembled at the aqueous interface between self-assembling peptides and polymer solutions have gained attention over the past decade. The goal of this mini-review is to highlight the advantages and challenges of utilizing polymer/peptide self-assembled macroscopic membranes toward the development of novel platforms for the study of the interaction of different cells with their environment. As most of the membranes features are determined as soon as they assemble, this review begins with a detailed description of the membranes formation mechanism, which evolved and developed over the years. The variety of membranes formed using different peptides and polymers is then described, with special focus placed on their unique structure. Then the bio-functions of the membranes and their emerging potential for bio-applications are presented. In the final section, the shortcomings preventing an extended use of these membranes as well as their future prospects, are discussed. This mini-review aims to give light to an emerging niche of biomaterials capable of forming hierarchical structures utilizing natural building blocks and the naturally occurring process of self-assembly, providing an innovative approach for creating scaffolds that mimic the natural extracellular environment.

Mixtures of nanometric rods and spherical micelles that are dominated by excluded volume (entropic) interactions tend to de-mix into sphere- and rod-rich mesophases. Here we report the observation of hybrid mesophases formed by mixtures of polymer-decorated sulfuric-acid-hydrolyzed cellulose nanocrystals (CNCs, rod-like particles), at high CNCs concentrations (7 wt%) where the native CNCs suspensions are at the bi-phasic (isotropic-chiral nematic) regime, and spherical micelles of the tri-block-copolymers Pluronic F127 (and F108) in aqueous suspension. The polymers adsorb onto the CNCs particles, and at F127 concentrations above (10-18 wt%) the macroscopic phases of the hybrid mixture are transformed into mesoscopic droplets with a nanostructure that differs from that of the CNCs or the native Pluronics, as indicated by small-angle X-ray scattering. The observed phases preserve the chiral nematic nature of the CNCs phases with a shortened pitch, suggesting that co-assembly of CNCs and self-assembling polymers may be used for engineering of emergent phases with emergent structures and properties.

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.