Supported Supramolecular Hydrogel Nanoarchitectonics for Tunable Biocatalytic Flow Activity.

Enzymatically-active polyelectrolyte multilayers containing n layers of phosphatase (APn-PEM) induce the formation of supported biocatalytic supramolecular hydrogels when brought in contact with the precursor tripeptide Fmoc-FFpY (Fmoc = N-fluorenylmethyloxycarbonyl; F = Phenylalanine; Y = Tyrosine; p = phosphate group). APn-PEM triggers the spatially-localized hydrogelation reaching 2, 17 and 350 µm of thickness for n = 1, 2 and 3, respectively. As observed by cryo scanning electron microscopy, a dense nanofibrous network underpinning the hydrogel shows parallelly orientated Fmoc-FFY peptide-based fibrils, perpendicular to the substrate. For the gel generated by the AP3-PEM, fluorescence confocal microscopy images show that during the peptide self-assembly, some enzymes are distributed in the hydrogel, preferentially located in few dozens of micrometers above the substrate. In addition, a self-assembly growth rate of 5 µm min-1 is determined when the hydrogelation starts. Through transmission electron microscopy immuno-labelling experiments on self-assemblies generated in solution, we observe that AP are decorating the Fmoc-FFY nanofibers. It is observed both a long-term stability and a higher biocatalytic activity of the so AP-encapsulated hydrogel compared to the bare APn-PEM. This bioactivity can be tuned by the number n in batch and under continuous flow conditions. To illustrate the versatility of this enzyme-supported strategy, multi-catalytic transformations in continuous flow conditions have been successfully carried out using supported supramolecular hydrogel.

Model to rationalize and predict the formation of organic patterns originating from an enzyme-assisted self-assembly Liesegang-like process of peptides in a host hydrogel.

Recently, we have investigated the enzyme-assisted self-assembly of precursor peptides diffusing in an enzyme-containing host gel, leading to various self-assembly profiles within the gel. At high enzyme concentrations, the reaction-diffusion self-assembly processes result in the formation of a continuous non-monotonous peptide self-assembly profile. At low enzyme concentrations, they result in the formation of individual self-assembled peptide microglobules and at intermediate enzyme concentrations both kinds of self-assembled structures coexist. Herein, we develop a Liesegang-type model that considers four major points: (i) the diffusion of the precursor peptides within the host gel, (ii) the diffusion of the enzymes in the gel, (iii) the enzymatic transformation of the precursor peptides into the self-assembling ones and (iv) the nucleation of these building blocks as the starting point of the self-assembly process. This process is treated stochastically. Our model predicts most of the experimentally observed features and in particular (i) the transition from a continuous to a microglobular pattern of self-assembled peptides through five types of patterns by decreasing the enzyme concentration in the host hydrogel. (ii) It also predicts that when the precursor peptide concentration decreases, the enzyme concentration at which the continuous/microglobules transition appears increases. (iii) Finally, it predicts that for peptides whose critical self-assembly concentration in solution decreases, the peptide concentration at which the continuous-to-microglobular transition decreases too. All these predictions are observed experimentally.

Mechanistic Insights into Hyaluronic Acid Induced Peptide Nanofiber Organization in Supramolecular Hydrogels.

Composite hydrogels composed of low-molecular-weight peptide self-assemblies and polysaccharides are gaining great interest as new types of biomaterials. Interactions between polysaccharides and peptide self-assemblies are well reported, but a molecular picture of their impact on the resulting material is still missing. Using the phosphorylated tripeptide precursor Fmoc-FFpY (Fmoc, fluorenylmethyloxycarbonyl; F, phenylalanine; Y, tyrosine; p, phosphate group), we investigated how hyaluronic acid (HA) influences the enzyme-assisted self-assembly of Fmoc-FFY generated in situ in the presence of alkaline phosphatase (AP). In the absence of HA, Fmoc-FFY peptides are known to self-assemble in nanometer thick and micrometer long fibers. The presence of HA leads to the spontaneous formation of bundles of several micrometers thickness. Using fluorescence recovery after photobleaching (FRAP), we find that in the bundles both (i) HA colocalizes with the peptide self-assemblies and (ii) its presence in the bundles is highly dynamic. The attractive interaction between negatively charged peptide fibers and negatively charged HA chains is explained through molecular dynamic simulations that show the existence of hydrogen bonds. Whereas the Fmoc-FFY peptide self-assembly itself is not affected by the presence of HA, this polysaccharide organizes the peptide nanofibers in a nematic phase visible by small-angle X-ray scattering (SAXS). The mean distance d between the nanofibers decreases by increasing the HA concentration c, but remains always larger than the diameter of the peptide nanofibers, indicating that they do not interact directly with each other. At a high enough HA concentration, the nematic organization transforms into an ordered 2D hexagonal columnar phase with a nanofiber distance d of 117 Å. Depletion interaction generated by the polysaccharides can explain the experimental power law variation d∼c-1/4 and is responsible for the bundle formation and organization. Such behavior is thus suggested for the first time on nano-objects using polymers partially adsorbing on self-assembled peptide nanofibers.

Validation of Milner's visco-elastic theory of sintering for the generation of porous polymers with finely tuned morphology.

Sacrificial sphere templating has become a method of choice to generate macro-porous materials with well-defined, interconnected pores. For this purpose, the interstices of a sphere packing are filled with a solidifying matrix, from which the spheres are subsequently removed to obtain interconnected voids. In order to control the size of the interconnections, viscous sintering of the initial sphere template has proven a reliable approach. To predict how the interconnections evolve with different sintering parameters, such as time or temperature, Frenkel's model has been used with reasonable success over the last 70 years. However, numerous investigations have shown that the often complex flow behaviour of the spheres needs to be taken into account. To this end, S. Milner [arXiv:1907.05862] developed recently a theoretical model which improves on some key assumptions made in Frenkel's model, leading to a slightly different scaling. He also extended this new model to take into account the visco-elastic response of the spheres. Using an in-depth investigation of templates of paraffin spheres, we provide here the first systematic comparison with Milner's theory. Firstly, we show that his new scaling describes the experimental data slightly better than Frenkel's scaling. We then show that the visco-elastic version of his model provides a significantly improved description of the data over a wide parameter range. We finally use the obtained sphere templates to produce macro-porous polyurethanes with finely controlled pore and interconnection sizes. The general applicability of Milner's theory makes it transferable to a wide range of formulations, provided the flow properties of the sphere material can be quantified. It therefore provides a powerful tool to guide the creation of sphere packings and porous materials with finely controlled morphologies.

Reversible Soft Mechanochemical Control of Biaryl Conformations through Crosslinking in a 3D Macromolecular Network.

Tuning the dihedral angle (DA) of axially chiral compounds can impact biological activity, catalyst efficiency, molecular motor performance, or chiroptical properties. Herein, we report gradual, controlled, and reversible changes in molecular conformation of a covalently linked binaphthyl moiety within a 3D polymeric network by application of a macroscopic stretching force. We managed direct observation of DA changes by measuring the circular dichroism signal of an optically pure BINOL-crosslinked elastomer network. Stretching the elastomer resulted in a widening of the DA between naphthyl rings when the BINOL was doubly grafted to the elastomer network; no effect was observed when a single naphthyl ring of the BINOL was grafted to the elastomer network. We have determined that ca. 170 % extension of the elastomers led to the transfer of a mechanical force to the BINOL moiety of 2.5 kcal mol-1 Å-1 (ca. 175 pN) in magnitude and results in the opening of the DA of BINOL up to 130°.

Autonomous Growth of a Spatially Localized Supramolecular Hydrogel with Autocatalytic Ability.

Autocatalysis and self-assembly are key processes in developmental biology and are involved in the emergence of life. In the last decade both of these features were extensively investigated by chemists with the final goal to design synthetic living systems. Herein, we describe the autonomous growth of a self-assembled soft material, that is, a supramolecular hydrogel, able to sustain its own formation through an autocatalytic mechanism that is not based on any template effect and emerges from a peptide (hydrogelator) self-assembly. A domino sequence of events starts from an enzymatically triggered peptide generation followed by self-assembly into catalytic nanofibers that induce and amplify their production over time, resulting in a 3D hydrogel network. A cascade is initiated by traces (10-18 m) of a trigger enzyme, which can be localized allowing for a spatial resolution of this autocatalytic buildup of hydrogel growth, an essential condition on the route towards further cell-mimic designs.

Phase Separation in Supramolecular Hydrogels Based on Peptide Self-Assembly from Enzyme-Coated Nanoparticles.

Spatial localization of biocatalysts, such as enzymes, has recently proven to be an effective process to direct supramolecular self-assemblies in a spatiotemporal way. In this work, silica nanoparticles (NPs) functionalized covalently by alkaline phosphatase (NPs@AP) induce the localized growth of self-assembled peptide nanofibers from NPs by dephosphorylation of Fmoc-FFpY peptides (Fmoc: fluorenylmethyloxycarbonyl; F: phenylalanine; Y: tyrosine; p: phosphate group). The fibrillary nanoarchitecture around NPs@AP underpins a homogeneous hydrogel, which unexpectedly undergoes a macroscopic shape change over time. This macroscopic change is due to a phase separation leading to a dense phase (in NPs and nanofibers) in the center of the vial and surrounded by a dilute one, which still contains NPs and peptide self-assemblies. We thus hypothesize that the phase separation is not a syneresis process. Such a change is only observed when the enzymes are localized on the NPs. The dense phase contracts with time until reaching a constant volume after several days. For a given phosphorylated peptide concentration, the dense phase contracts faster when the NPs@AP concentration is increased. For a given NPs@AP concentration, it condenses faster when the peptide concentration increases. We hypothesize that the appearance of a dense phase is not only due to attractive interactions between NPs@AP but also to the strong interactions of self-assembled peptide nanofibers with the enzymes, covalently fixed on the NPs.

Supported Catalytically Active Supramolecular Hydrogels for Continuous Flow Chemistry.

Inspired by biology, one current goal in supramolecular chemistry is to control the emergence of new functionalities arising from the self-assembly of molecules. In particular, some peptides can self-assemble and generate exceptionally catalytically active fibrous networks able to underpin hydrogels. Unfortunately, the mechanical fragility of these materials is incompatible with process developments, relaying this exciting field to academic curiosity. Here, we show that this drawback can be circumvented by enzyme-assisted self-assembly of peptides initiated at the walls of a supporting porous material. We applied this strategy to grow an esterase-like catalytically active supramolecular hydrogel (CASH) in an open-cell polymer foam, filling the whole interior space. Our supported CASH material is highly efficient towards inactivated esters and enables the kinetic resolution of racemates. This hybrid material is robust enough to be used in continuous flow reactors, and is reusable and stable over months.

Mimicking the Chemistry of Natural Eumelanin Synthesis: The KE Sequence in Polypeptides and in Proteins Allows for a Specific Control of Nanosized Functional Polydopamine Formation.

The oxidation of dopamine and of other catecholamines leads to the formation of conformal films on the surface of all known materials and to the formation of a precipitate in solution. In some cases, it has been shown that the addition of additives in the dopamine solution, like certain surfactants or polymers, polyelectrolytes, and certain proteins, allows to get polydopamine nanoparticles of controlled size and the concomitant decrease, in an additive/dopamine dependent manner, in film formation on the surface of the reaction beaker. However, the mechanism behind this controlled oxidation and self-assembly of catecholamines is not known. In this article, it is shown that a specific diad of amino acids in proteins, namely KE, allows for specific control in the oxidation-self-assembly of dopamine to obtain polydopamine@protein core-shell nanoparticles which are biocompatible. The interactions between dopamine and the adjacent KE amino acids potentially responsible for the size control of polydopamine aggregates was investigated by molecular dynamics simulations. The obtained core-shell nanoparticles display the biological activity of the protein used to control the self-assembly of PDA. The photon to heat conversion ability of PDA is conserved in the PDA@protein particles.

Surface-Assisted Self-Assembly Strategies Leading to Supramolecular Hydrogels.

Localized molecular self-assembly processes leading to the growth of nanostructures exclusively from the surface of a material is one of the great challenges in surface chemistry. In the last decade, several works have been reported on the ability of modified or unmodified surfaces to manage the self-assembly of low-molecular-weight hydrogelators (LMWH) resulting in localized supramolecular hydrogel coatings mainly based on nanofiber architectures. This Minireview highlights all strategies that have emerged recently to initiate and localize LMWH supramolecular hydrogel formation, their related fundamental issues and applications.

Control of Surface-Localized, Enzyme-Assisted Self-Assembly of Peptides through Catalyzed Oligomerization.

Localized self-assembly allowing both spatial and temporal control over the assembly process is essential in many biological systems. This can be achieved through localized enzyme-assisted self-assembly (LEASA), also called enzyme-instructed self-assembly, where enzymes present on a substrate catalyze a reaction that transforms noninteracting species into self-assembling ones. Very few LEASA systems have been reported so far, and the control of the self-assembly process through the surface properties represents one essential step toward their use, for example, in artificial cell mimicry. Here, we describe a new type of LEASA system based on α-chymotrypsin adsorbed on a surface, which catalyzes the production of (KL)nOEt oligopeptides from a KLOEt (K: lysine; L: leucine; OEt ethyl ester) solution. When a critical concentration of the formed oligopeptides is reached near the surface, they self-assemble into ß-sheets resulting in a fibrillar network localized at the interface that can extend over several micrometers. One significant feature of this process is the existence of a lag time before the self-assembly process starts. We investigate, in particular, the effect of the α-chymotrypsin surface density and KLOEt concentration on the self-assembly kinetics. We find that the lag time can be finely tuned through the surface density in α-chymotrypsin and KLOEt concentration. For a given surface enzyme concentration, a critical KLOEt concentration exists below which no self-assembly takes place. This concentration increases when the surface density in enzyme decreases.

Localized Supramolecular Peptide Self-Assembly Directed by Enzyme-Induced Proton Gradients.

Electrodes are ideal substrates for surface localized self-assembly processes. Spatiotemporal control over such processes is generally directed through the release of ions generated by redox reactions occurring specifically at the electrode. The so-used gradients of ions proved their effectiveness over the last decade but are in essence limited to material-based electrodes, considerably reducing the scope of applications. Herein is described a strategy to enzymatically generate proton gradients from non-conductive surfaces. In the presence of oxygen, immobilization of glucose oxidase (GOx) on a multilayer film provides a flow of protons through enzymatic oxidation of glucose by GOx. The confined acidic environment located at the solid-liquid interface allows the self-assembly of Fmoc-AA-OH (Fmoc=fluorenylmethyloxycarbonyl and A=alanine) dipeptides into ß-sheet nanofibers exclusively from and near the surface. In the absence of oxygen, a multilayer nanoreactor containing GOx and horseradish peroxidase (HRP) similarly induces Fmoc-AA-OH self-assembly.

Soft-Mechanochemistry: Mechanochemistry Inspired by Nature.

Cells and bacteria use mechanotransduction processes to transform a mechanical force into a chemical/biochemical response. The area of chemistry where chemical reactions are induced by mechanical forces is called mechanochemistry. Over the last few years, chemists developed force-induced reactions affecting covalent bonds in molecules under tension which requires high energy input and/or high intensity forces. In contrast, in nature, mechanotransduction processes take place with forces of much weaker intensity and much less demanding energy. They are mainly based on protein conformational changes or changes in supramacromolecular architectures. Mechanochemistry based on such low-energy-demanding processes and which does not affect chemical bonds can be called soft-mechanochemistry. In this feature article, we first discuss some examples of soft-mechanochemistry processes encountered in nature, in particular, cryptic sites, allowing us to define more precisely the concepts underlying soft-mechanochemistry. A series of examples, developed over the past few years, of chemomechanoresponsive systems based on soft-mechanochemistry principles are given. We describe, in particular, cryptic site surfaces, enzymatically active films whose activity can be modulated by stretching and films where stretching induces changes in their fluorescence properties. Finally, we give our view of the future of soft-mechanochemistry.

Immunomodulation with Self-Crosslinked Polyelectrolyte Multilayer-Based Coatings.

This study aims to design an optimal polyelectrolyte multilayer film of poly-l-lysine (PLL) and hyaluronic acid (HA) as an anti-inflammatory cytokine release system in order to decrease the implant failure due to any immune reactions. The chemical modification of the HA with aldehyde moieties allows self-cross-linking of the film and an improvement in the mechanical properties of the film. The cross-linking of the film and the release of immunomodulatory cytokine (IL-4) stimulate the differentiation of primary human monocytes seeded on the films into pro-healing macrophages phenotype. This induces the production of anti-inflammatory cytokines (IL1-RA and CCL18) and the decrease of pro-inflammatory cytokines secreted (IL-12, TNF-α, and IL-1ß). Moreover, we demonstrate that cross-linking PLL/HA film using HA-aldehyde is already effective by itself to limit inflammatory processes. Finally, this functionalized self-cross-linked PLL/HA-aldehyde films constitutes an innovative and efficient candidate for immunomodulation of any kind of implants of various architecture and properties.

Selective Nanotrench Filling by One-Pot Electroclick Self-Constructed Nanoparticle Films.

Integration of nanoparticles (NPs) into nanodevices is a challenge for enhanced sensor development. Using NPs as building blocks, a bottom-up approach based on one-pot morphogen-driven electroclick chemistry is reported to self-construct dense and robust conductive Fe3O4 NP films. Deposited covalent NP assemblies establish an electrical connection between two gold electrodes separated by a 100 nm-wide nanotrench.

Polyelectrolyte Multilayers: A Versatile Tool for Preparing Antimicrobial Coatings.

The prevention of pathogen colonization of medical implants represents a major medical and financial issue. The development of antimicrobial coatings aimed at protecting against such infections has thus become a major field of scientific and technological research. Three main strategies are developed to design such coatings: (i) the prevention of microorganisms adhesion and the killing of microorganisms (ii) by contact and (iii) by the release of active compounds in the vicinity of the implant. Polyelectrolyte multilayer (PEM) technology alone covers the entire widespread spectrum of functionalization possibilities. PEMs are obtained through the alternating deposition of polyanions and polycations on a substrate, and the great advantages of PEMs are that (i) they can be applied to almost any type of substrate whatever its shape and composition; (ii) various chemical, physicochemical, and mechanical properties of the coatings can be obtained; and (iii) active compounds can be embedded and released in a controlled manner. In this article we will give an overview of the field of PEMs applied to the design of antimicrobial coatings, illustrating the large versatility of the PEM technology.

Stable Bioactive Enzyme-Containing Multilayer Films Based on Covalent Cross-Linking from Mussel-Inspired Adhesives.

The use of immobilized enzymes is mandatory for the easy separation of the enzyme, the unreacted substrates, and the obtained products to allow repeated enzymatic assays without cumbersome purification steps. The immobilization procedure is however critical to obtain a high fraction of active enzyme. In this article, we present an enzyme immobilization strategy based on a catechol functionalized alginate. We demonstrate that alkaline phosphatase (ALP) remains active in multilayered films made with alginate modified with catechol moieties (AlgCat) for long duration, that is, up to 7 weeks, provided the multilayered architecture is cross-linked with sodium periodate. This cross-linking reaction allows to create covalent bonds between the amino groups of ALP and the quinone group carried by the modified alginate. In the absence of cross-linking, the enzymatic activity is rapidly lost and this reduction is mainly due to enzyme desorption. We also show that NaIO4 cross-linked (AlgCat-Alp)n films can be freeze-dried and reused at least 3 weeks later without lost in enzymatic activity.

Morphogen Electrochemically Triggered Self-Construction of Polymeric Films Based on Mussel-Inspired Chemistry.

Inspired by the strong chemical adhesion mechanism of mussels, we designed a catechol-based electrochemically triggered self-assembly of films based on ethylene glycol molecules bearing catechol groups on both sides and denoted as bis-catechol molecules. These molecules play the role of morphogens and, in contrast to previously investigated systems, they are also one of the constituents, after reaction, of the film. Unable to interact together, commercially available poly(allylamine hydrochloride) (PAH) chains and bis-catechol molecules are mixed in an aqueous solution and brought in contact with an electrode. By application of defined potential cycles, bis-catechol molecules undergo oxidation leading to molecules bearing "reactive" quinone groups which diffuse toward the solution. In this active state, the quinones react with amino groups of PAH through Michael addition and Schiff's base condensation reaction. The application of cyclic voltammetry (CV) between 0 and 500 mV (vs Ag/AgCl, scan rate of 50 mV/s) of a PAH/bis-catechol solution results in a fast self-construction of a film that reaches a thickness of 40 nm after 60 min. The films present a spiky structure which is attributed to the use of bis-functionalized molecules as one component of the films. XPS measurements show the presence of both PAH and bis-catechol cross-linked together in a covalent way. We show that the amine/catechol ratio is an important parameter which governs the film buildup. For a given amine/catechol ratio, it does exist an optimum CV scan rate leading to a maximum of the film thickness as a function of the scan rate.

Film Self-Assembly of Oppositely Charged Macromolecules Triggered by Electrochemistry through a Morphogenic Approach.

The development of new surface functionalization methods that are easy to use, versatile, and allow local deposition represents a real scientific challenge. Overcoming this challenge, we present here a one-pot process that consists in self-assembling, by electrochemistry on an electrode, films made of oppositely charged macromolecules. This method relies on a charge-shifting polyanion, dimethylmaleic-modified poly(allylamine) (PAHd), that undergoes hydrolysis at acidic pH, leading to an overall switching of its charge. When a mixture of the two polyanions, PAHd and poly(styrenesulfonate) (PSS), is placed in contact with an electrode, where the pH is decreased locally by electrochemistry, the transformation of PAHd into a polycation (PAH) leads to the continuous self-assembly of a nanometric PAH/PSS film by electrostatic interactions. The pH decrease is obtained by the electrochemical oxidation of hydroquinone, which produces protons locally over nanometric distances. Using a negatively charged enzyme, alkaline phosphatase (AP), instead of PSS, this one-pot process allows the creation of enzymatically active films. Under mild conditions, self-assembled PAH/AP films have an enzymatic activity which is adjustable simply by controlling the self-assembly time. The selective functionalization of microelectrode arrays by PAH/AP was achieved, opening the route toward miniaturized biosensors.

Bioactive Seed Layer for Surface-Confined Self-Assembly of Peptides.

The design and control of molecular systems that self-assemble spontaneously and exclusively at or near an interface represents a real scientific challenge. We present here a new concept, an active seed layer that allows to overcome this challenge. It is based on enzyme-assisted self-assembly. An enzyme, alkaline phosphatase, which transforms an original peptide, Fmoc-FFY(PO4 (2-) ), into an efficient gelation agent by dephosphorylation, is embedded in a polyelectrolyte multilayer and constitutes the "reaction motor". A seed layer composed of a polyelectrolyte covalently modified by anchoring hydrogelator peptides constitutes the top of the multilayer. This layer is the nucleation site for the Fmoc-FFY peptide self-assembly. When such a film is brought in contact with a Fmoc-FFY(PO4 (2-) ) solution, a nanofiber network starts to form almost instantaneously which extents up to several micrometers into the solution after several hours. We demonstrate that the active seed layer allows convenient control over the self-assembly kinetics and the geometric features of the fiber network simply by changing its peptide density.