Structure-Property Relationships of Granular Hybrid Hydrogels Formed through Polyelectrolyte Complexation.

Hybrid hydrogels are hydrogels that exhibit heterogeneity in the network architecture by means of chemical composition and/or microstructure. The different types of interactions, together with structural heterogeneity, which can be created on different length scales, determine the mechanical properties of the final material to a large extent. In this work, the microstructure-mechanical property relationships for a hybrid hydrogel that contains both electrostatic and covalent interactions are investigated. The hybrid hydrogel is composed of a microphase-separated polyelectrolyte complex network (PEC) made of poly(4-styrenesulfonate) and poly(diallyldimethylammonium chloride) within a soft and elastic polyacrylamide hydrogel network. The system exhibits a granular structure, which is attributed to the liquid-liquid phase separation into complex coacervate droplets induced by the polymerization and the subsequent crowding effect of the polyacrylamide chains. The coacervate droplets are further hardened into PEC granules upon desalting the hydrogel. The structure formation is confirmed by a combination of electron microscopic imaging and molecular dynamics simulations. The interpenetration of both networks is shown to enhance the toughness of the resulting hydrogels due to the dissipative behavior of the PEC through the rupture of electrostatic interactions. Upon cyclic loading-unloading, the hydrogels show recovery of up to 80% of their original dissipative behavior in less than 300 s of rest with limited plasticity. The granular architecture and the tough and self-recoverable properties of the designed hybrid networks make them good candidates for applications, such as shape-memory materials, actuators, biological tissue mimics, and elastic substrates for soft sensors.

Effect of Polyelectrolyte Charge Density on the Linear Viscoelastic Behavior and Processing of Complex Coacervate Adhesives.

It is well-known that the phase behavior and physicochemical and adhesive properties of complex coacervates are readily tuneable with the salt concentration of the medium. For toxicity reasons, however, the maximum applicable salt concentration in biomedical applications is typically low. Consequently, other strategies must be implemented in order to optimize the properties of the resulting complex coacervates. In this work, the effect of the charge density of a strong polyanion on the properties of complex coacervates was studied. To control this charge density, statistical anionic/charge-neutral hydrophilic copolymers were synthesized by means of an elegant protection/deprotection strategy and subsequently complexed with a strong polycation. The resulting complexes were observed to have an increasing water content as well as faster relaxation dynamics, with either increasing salt concentration or decreasing charge density. Time-salt and time-salt-charge density superpositions could be performed and showed that the relaxation mechanism of the complex coacervates remained unchanged. When the charge density was decreased, lower salt concentration complexes became suitable for viscoelastic adhesion with improved injectability. Such complex coacervates are promising candidates for injectable biomedical adhesives.

Hydrophobically modified complex coacervates for designing aqueous pressure-sensitive adhesives.

The rheology of complex coacervates can be elegantly tuned via the design and control of specific non-covalent hydrophobic interactions between the complexed polymer chains. The well-controlled balance between elasticity and energy dissipation makes complex coacervates perfect candidates for pressure-sensitive adhesives (PSAs). In this work, the polyanion poly(3-sulfopropyl methacrylate) (PSPMA) and the polycation quaternized poly(4-vinylpyridine) (QP4VP) were used to prepare complex coacervates in water. Progressive increase of hydrophobicity is introduced to the polyanion via partial deprotection of the protected precursor. Hence, the polymer chains in the complex coacervates can interact via both electrostatic (controlled by the amount of salt) and hydrophobic (controlled by the deprotection degree) interactions. It was observed that: (i) a rheological time-salt-hydrophobicity superposition principle is applicable, and can be used as a predictive tool for rheology, (ii) the slowdown of the stress relaxation dynamics, due to the increase of hydrophobic stickers (lower deprotection degree), can be captured by the sticky-Rouse model, and (iii) the systematic variation of hydrophobic stickers, amount of salt, and molecular weight of the polymers, enables the identification of optimizing parameters to design aqueous PSA systems. The presented results offer new pathways to control the rheology of complex coacervates and their applicability as PSAs.

Toward Effective and Adsorption-Based Antifouling Zipper Brushes: Effect of pH, Salt, and Polymer Design.

The undesired spontaneous deposition and accumulation of matter on surfaces, better known as fouling, is a problematic and often inevitable process plaguing a variety of industries. This detrimental process can be reduced or even prevented by coating surfaces with a dense layer of end-grafted polymer: a polymer brush. Producing such polymer brushes via adsorption presents a very attractive technique, as large surfaces can be coated in a quick and simple manner. Recently, we introduced a simple and scalable two-step adsorption strategy to fabricate block copolymer-based antifouling coatings on hydrophobic surfaces. This two-step approach involved the initial adsorption of hydrophobic-charged diblock copolymer micelles acting as a primer, followed by the complexation of oppositely charged-antifouling diblock copolymers to form the antifouling brush coating. Here, we significantly improve this adsorption-based zipper brush via systematic tuning of various parameters, including pH, salt concentration, and polymer design. This study reveals several key outcomes. First of all, increasing the hydrophobic/hydrophilic block ratio of the anchoring polymeric micelles (i.e., decreasing the hydrophilic corona) promotes adsorption to the surface, resulting in the most densely packed, uniform, and hydrophilic primer layers. Second, around a neutral pH and at a low salt concentration (1 mM), complexation of the weak polyelectrolyte (PE) blocks results in brushes with the best antifouling efficacy. Moreover, by tuning the ratio between these PE blocks, the brush density can be increased, which is also directly correlated to the antifouling performance. Finally, switching to different antifouling blocks can increase the internal density or strengthen the bound hydration layer of the brush, leading to an additional enhancement of the antifouling properties (>99% lysozyme, 87% bovine serum albumin).

Effect of Dynamically Arrested Domains on the Phase Behavior, Linear Viscoelasticity and Microstructure of Hyaluronic Acid - Chitosan Complex Coacervates.

Complex coacervates make up a class of versatile materials formed as a result of the electrostatic associations between oppositely charged polyelectrolytes. It is well-known that the viscoelastic properties of these materials can be easily altered with the ionic strength of the medium, resulting in a range of materials from free-flowing liquids to gel-like solids. However, in addition to electrostatics, several other noncovalent interactions could influence the formation of the coacervate phase depending on the chemical nature of the polymers involved. Here, the importance of intermolecular hydrogen bonds on the phase behavior, microstructure, and viscoelasticity of hyaluronic acid (HA)-chitosan (CHI) complex coacervates is revealed. The density of intermolecular hydrogen bonds between CHI units increases with increasing pH of coacervation, which results in dynamically arrested regions within the complex coacervate, leading to elastic gel-like behavior. This pH-dependent behavior may be very relevant for the controlled solidification of complex coacervates and thus for polyelectrolyte material design.

One-Pot Synthesis of Strong Anionic/Charge-Neutral Amphiphilic Block Copolymers.

Despite the ever more versatile polymerization techniques that are becoming available, the synthesis of macromolecules with tailored functionalities can remain a lengthy endeavor. This becomes more conspicuous when the implementation of incompatible chemistries (i.e., strong polyelectrolytes) within sequence-controlled polymers is desired, often requiring (i) polymerization, (ii) chain extension, and (iii) postpolymerization modification. Herein, we explore the production of strong anionic/charge-neutral block copolymers (BCPs) in a one-pot fashion. This straightforward three-step process includes the synthesis of a macroinitiator and chain extension via rapid and efficient photomediated atom transfer radical polymerization, followed by in situ deprotection to expose the polyanionic domains. The resulting BCPs, which are strong amphiphiles by nature, are capable of self-assembly in aqueous media, as evidenced by dynamic light scattering, small-angle X-ray scattering, ζ-potential measurements, and transmission electron microscopy. We further demonstrate the versatility of our methodology by producing several BCPs through sampling of a single reaction mixture, enabling the straightforward production of strong polymer amphiphiles.

Electrically Conductive and Highly Stretchable Piezoresistive Polymer Nanocomposites via Oxidative Chemical Vapor Deposition.

Electrically conductive polymer nanocomposites have been the subject of intense research due to their promising potential as piezoresistive biomedical sensors, leveraging their flexibility and biocompatibility. Although intrinsically conductive polymers such as polypyrrole (PPy) and polyaniline have emerged as lucrative candidates, they are extremely limited in their processability by conventional solution-based approaches. In this work, ultrathin nanostructured coatings of doped PPy are realized on polyurethane films of different architectures via oxidative chemical vapor deposition to develop stretchable and flexible resistance-based strain sensors. Holding the substrates perpendicular to the reactant flows facilitates diffusive transport and ensures excellent conformality of the interfacial integrated PPy coatings throughout the 3D porous electrospun fiber mats in a single step. This allows the mechanically robust (stretchability > 400%, with fatigue resistance up to 1000 cycles) nanocomposites to elicit a reversible change of electrical resistance when subjected to consecutive cycles of stretching and releasing. The repeatable performance of the strain sensor is linear due to dimensional changes of the conductive network in the low-strain regime (ε ≤ 50%), while the evolution of nano-cracks leads to an exponential increase, which is observed in the high-strain regime, recording a gauge factor as high as 46 at 202% elongational strain. The stretchable conductive polymer nanocomposites also show biocompatibility toward human dermal fibroblasts, thus providing a promising path for use as piezoresistive strain sensors and finding applications in biomedical applications such as wearable, skin-mountable flexible electronics.

Scalable Fabrication of Reversible Antifouling Block Copolymer Coatings via Adsorption Strategies.

Fouling remains a widespread challenge as its nonspecific and uncontrollable character limits the performance of materials and devices in numerous applications. Although many promising antifouling coatings have been developed to reduce or even prevent this undesirable adhesion process, most of them suffer from serious limitations, specifically in scalability. Whereas scalability can be particularly problematic for covalently bound antifouling polymer coatings, replacement by physisorbed systems remains complicated as it often results in less effective, low-density films. In this work, we introduce a two-step adsorption strategy to fabricate high-density block copolymer-based antifouling coatings on hydrophobic surfaces, which exhibit superior properties compared to one-step adsorbed coatings. The obtained hybrid coating manages to effectively suppress the attachment of both lysozyme and bovine serum albumin, which can be explained by its dense and homogeneous surface structure as well as the desired polymer conformation. In addition, the intrinsic reversibility of the adhered complex coacervate core micelles allows for the successful triggered release and regeneration of the hybrid coating, resulting in full recovery of its antifouling properties. The simplicity and reversibility make this a unique and promising antifouling strategy for large-scale underwater applications.

Bioinspired Processing: Complex Coacervates as Versatile Inks for 3D Bioprinting.

3D bioprinting is a powerful fabrication technique in biomedical engineering, which is currently limited by the number of available materials that meet all physicochemical and cytocompatibility requirements for biomaterial inks. Inspired by the key role of coacervation in the extrusion and spinning of many natural materials, hyaluronic acid-chitosan complex coacervates are proposed here as tunable biomaterial inks. Complex coacervates are obtained through an associative liquid-liquid phase separation driven by electrostatic attraction between oppositely charged macromolecules. They offer bioactive properties and facile modulation of their mechanical properties through mild physicochemical changes in the environment, making them attractive for 3D bioprinting. Fine-tuning the salt concentration, pH, and molecular weight of the constituent polymers results in biomaterial inks that are printable in air and water. The biomaterial ink, initially a viscoelastic fluid, transitions into a viscoelastic solid upon printing due to dehydration (for printing in air) or due to a change in pH and ionic composition (for printing in solution). Consequently, scaffolds printed using the complex coacervate inks are stable without the need for post-printing processing. Fabricated cell culture scaffolds are cytocompatible and show long-term topological stability. These results pave the way to a new class of easy-to-handle tunable biomaterials for biofabrication.

Bioinspired Processing of Keratin into Upcycled Fibers through pH-Induced Coacervation.

Keratin is an important byproduct of the animal industry, but almost all of it ends up in landfills due to a lack of efficient recycling methods. To make better use of keratin-based natural resources, the current extraction and processing strategies need to be improved or replaced by more sustainable and cost-effective processes. Here, we developed a simple and environmentally benign method to process extracted keratin, using HCl to induce the formation of a coacervate, a separate aqueous phase with a very high protein concentration. Remarkably, this pH-induced coacervation did not result in the denaturation of keratin, and we could even observe an increase in the amount of ordered secondary structures. The low-pH coacervates could be extruded and wet-spun into high-performance keratin fibers, without requiring heating or any organic solvents. The secondary structure of keratin was largely conserved in these regenerated fibers, which exhibited excellent mechanical performance. The process developed in this study represents a simple and environmentally friendly strategy to upcycle waste keratin into high-performance materials.

Water-Processable, Stretchable, and Ion-Conducting Coacervate Fibers from Keratin Associations with Polyelectrolytes.

Keratin is one of the most abundant biopolymers, produced on a scale of millions of tons per year but often simply discarded as waste. Due to its abundance, biocompatibility, and excellent mechanical properties, there is an extremely high interest in developing protocols for the recycling of keratin and its conversion into protein-based materials. In this work, we describe a novel protocol for the conversion of keratin from wool into hybrid fibers. Our protocol uses a synthetic polyanion, which undergoes complex coacervation with keratin, leading to a viscous liquid phase that can be used directly as a dope for dry-spinning. The use of polyelectrolyte complexation allows us to use all of the extracted keratin, unlike previous works that were limited to the fraction with the highest molecular weight. The fibers prepared by this protocol show excellent mechanical properties, humidity responsiveness, and ion conductivity, which makes them promising candidates for applications as a strain sensor.

Strong Anionic/Charge-Neutral Block Copolymers from Cu(0)-Mediated Reversible Deactivation Radical Polymerization.

Despite recent developments in controlled polymerization techniques, the straightforward synthesis of block copolymers that feature both strong anionic and charge-neutral segments remains a difficult endeavor. In particular, solubility issues may arise during the direct synthesis of strong amphiphiles and typical postpolymerization deprotection often requires harsh conditions. To overcome these challenges, we employed Cu(0)-mediated reversible deactivation radical polymerization (Cu(0)-RDRP) on a hydrophobic isobutoxy-protected 3-sulfopropyl acrylate. Cu(0)-RDRP enables the rapid synthesis of the polymer, reaching high conversions and low dispersities while using a single solvent system and low amounts of copper species. These macromolecules are straightforward to characterize and can subsequently be deprotected in a mild yet highly efficient fashion to expose their strongly charged nature. Furthermore, a protected sulfonate segment could be grown from a variety of charge-neutral macroinitiators to produce, after the use of the same deprotection chemistry, a library of amphiphilic, double-hydrophilic as well as thermoresponsive block copolymers (BCPs). The ability of these various BCPs to self-assemble in aqueous media was further studied by dynamic light scattering, ζ-potential measurements as well as atomic force and electron microscopy.

Ultra-sounding out a technique that sticks.

Ultrasound can be used to promote the physical interlocking of adhesives and tissues.

The Unfolded Protein Response Sensor PERK Mediates Stiffness-Dependent Adaptation in Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor in adults. In addition to genetic causes, the tumor microenvironment (TME), including stiffening of the extracellular matrix (ECM), is a main driver of GBM progression. Mechano-transduction and the unfolded protein response (UPR) are essential for tumor-cell adaptation to harsh TME conditions. Here, we studied the effect of a variable stiff ECM on the morphology and malignant properties of GBM stem cells (GSCs) and, moreover, examined the possible involvement of the UPR sensor PERK herein. For this, stiffness-tunable human blood plasma (HBP)/alginate hydrogels were generated to mimic ECM stiffening. GSCs showed stiffness-dependent adaptation characterized by elongated morphology, increased proliferation, and motility which was accompanied by F-Actin cytoskeletal remodeling. Interestingly, in PERK-deficient GSCs, stiffness adaptation was severely impaired, which was evidenced by low F-Actin levels, the absence of F-Actin remodeling, and decreased cell proliferation and migration. This impairment could be linked with Filamin-A (FLN-A) expression, a known interactor of PERK, which was strongly reduced in PERK-deficient GSCs. In conclusion, we identified a novel PERK/FLNA/F-Actin mechano-adaptive mechanism and found a new function for PERK in the cellular adaptation to ECM stiffening.

Protected Poly(3-sulfopropyl methacrylate) Copolymers: Synthesis, Stability, and Orthogonal Deprotection.

Because of their permanent charge, strong polyelectrolytes remain challenging to characterize, in particular, when they are combined with hydrophobic features. For this reason, they are typically prepared through a postmodification of a fully hydrophobic precursor. Unfortunately, these routes often result in an incomplete functionalization or otherwise require harsh reaction conditions, thus limiting their applicability. To overcome these problems, in this work a strategy is presented that facilitates the preparation of well-defined strong polyanions by starting from protected 3-sulfopropyl methacrylate monomers. Depending on the chemistry of the protecting group, the hydrophobic precursor could be quantitatively converted into a strong polyanion under nucleophilic, acidic, or basic conditions. As a proof of concept, orthogonally protected diblock copolymers were synthesized, selectively deprotected, and allowed to self-assemble in aqueous solution. Further conversion into a fully water-soluble polyanion was achieved by deprotecting the second block as well.

Physics of Brain Cancer: Multiscale Alterations of Glioblastoma Cells under Extracellular Matrix Stiffening.

The biology and physics underlying glioblastoma is not yet completely understood, resulting in the limited efficacy of current clinical therapy. Recent studies have indicated the importance of mechanical stress on the development and malignancy of cancer. Various types of mechanical stress activate adaptive tumor cell responses that include alterations in the extracellular matrix (ECM) which have an impact on tumor malignancy. In this review, we describe and discuss the current knowledge of the effects of ECM alterations and mechanical stress on GBM aggressiveness. Gradual changes in the brain ECM have been connected to the biological and physical alterations of GBM cells. For example, increased expression of several ECM components such as glycosaminoglycans (GAGs), hyaluronic acid (HA), proteoglycans and fibrous proteins result in stiffening of the brain ECM, which alters inter- and intracellular signaling activity. Several mechanosensing signaling pathways have been identified that orchestrate adaptive responses, such as Hippo/YAP, CD44, and actin skeleton signaling, which remodel the cytoskeleton and affect cellular properties such as cell-cell/ECM interactions, growth, and migration/invasion of GBM cells. In vitro, hydrogels are used as a model to mimic the stiffening of the brain ECM and reconstruct its mechanics, which we also discuss. Overall, we provide an overview of the tumor microenvironmental landscape of GBM with a focus on ECM stiffening and its associated adaptive cellular signaling pathways and their possible therapeutic exploitation.

Responsive Pickering Emulsions Stabilized by Frozen Complex Coacervate Core Micelles.

Frozen complex coacervate core micelles (C3Ms) were developed as a class of particle stabilizers for Pickering emulsions. The C3Ms are composed of a core of electrostatically interacting weak polyelectrolytes, poly(acrylic acid) (pAA) and poly(dimethylaminopropylacrylamide) (pDMAPAA), surrounded by a corona of water-soluble and surface active poly(N-isopropylacrylamide) (pNiPAM). Mixing parameters of the two polymer solutions, including pH, mixing method, charge ratio, and salinity of the medium, were carefully controlled, leading to monodisperse, colloidally stable C3Ms. A combination of dynamic light scattering and proton nuclear magnetic resonance experiments showed that the C3Ms gradually disassembled from a dynamically frozen core state in pure water into free polyelectrolyte chains above 0.8 M NaCl. Upon formulation of dodecane-in-water emulsions, the frozen C3Ms adsorb as particles at the droplet interfaces in striking contrast with most of the conventional micelles made of amphiphilic block copolymers which fall apart at the interface. Eventually, increasing the salt concentration of the system triggered disassembly of the C3Ms, which led to emulsion destabilization.

Enhanced stability of complex coacervate core micelles following different core-crosslinking strategies.

Complex coacervate core micelles (C3Ms) are formed by mixing aqueous solutions of a charged (bio)macromolecule with an oppositely charged-neutral hydrophilic diblock copolymer. The stability of these structures is dependent on the ionic strength of the solution; above a critical ionic strength, the micelles will completely disintegrate. This instability at high ionic strengths is the main drawback for their application in, e.g., drug delivery systems or protein protection. In addition, the stability of C3Ms composed of weak polyelectrolytes is pH-dependent as well. The aim of this study is to assess the effectiveness of covalent crosslinking of the complex coacervate core to improve the stability of C3Ms. We studied the formation of C3Ms using a quaternized and amine-functionalized cationic-neutral diblock copolymer, poly(2-vinylpyridine)-block-poly(ethylene oxide) (QP2VP-b-PEO), and an anionic homopolymer, poly(acrylic acid) (PAA). Two different core-crosslinking strategies were employed that resulted in crosslinks between both types of polyelectrolyte chains in the core (i.e., between QP2VP and PAA) or in crosslinks between polyelectrolyte chains of the same type only (i.e., QP2VP). For these two strategies we used the crosslinkers 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and dimethyl-3,3'-dithiopropionimidate dihydrochloride (DTBP), respectively. EDC provides permanent crosslinks, while DTBP crosslinks can be broken by a reducing agent. Dynamic light scattering showed that both approaches significantly improved the stability of C3Ms against salt and pH changes. Furthermore, reduction of the disulphide bridges in the DTBP core-crosslinked micelles largely restored the original salt-stability profile. Therefore, this feature provides an excellent starting point for the application of C3Ms in controlled release formulations.

Charged Polypeptide Tail Boosts the Salt Resistance of Enzyme-Containing Complex Coacervate Micelles.

Encapsulation of proteins can have advantages for their protection, stability, and delivery purposes. One of the options to encapsulate proteins is to incorporate them in complex coacervate core micelles (C3Ms). This can easily be achieved by mixing aqueous solutions of the protein and an oppositely charged neutral-hydrophilic diblock copolymer. However, protein-containing C3Ms often suffer from salt-inducible disintegration due to the low charge density of proteins. The aim of this study is to improve the salt stability of protein-containing C3Ms by increasing the net charge of the protein by tagging it with a charged polypeptide. As a model protein, we used CotA laccase and generated variants with 10, 20, 30, and 40 glutamic acids attached at the C-terminus of CotA using genetic engineering. Micelles were obtained by mixing the five CotA variants with poly(N-methyl-2-vinyl-pyridinium)-block-poly(ethylene oxide) (PM2VP128-b-PEO477) at pH 10.8. Hydrodynamic radii of the micelles of approximately 31, 27, and 23 nm for native CotA, CotA-E20, and CotA-E40, respectively, were determined using dynamic light scattering (DLS) and fluorescence correlation spectroscopy (FCS). The encapsulation efficiency was not affected using enzymes with a polyglutamic acid tail but resulted in more micelles with a smaller number of enzyme molecules per micelle. Furthermore, it was shown that the addition of a polyglutamic acid tail to CotA indeed resulted in improved salt stability of enzyme-containing C3Ms. Interestingly, the polyglutamic acid CotA variants showed an enhanced enzyme activity. This study demonstrates that increasing the net charge of enzymes through genetic engineering is a promising strategy to improve the practical applicability of C3Ms as enzyme delivery systems.

Hooked on mushrooms: Preparation and mechanics of a bioinspired soft probabilistic fastener.

Probabilistic fasteners are known to provide strong attachment onto their respective surfaces. Examples are Velcro® and the "3M dual lock" system. However, these systems typically only function using specific counter surfaces and are often destructive to other surfaces such as fabrics. Moreover, the design parameters to optimize their functionality are not obvious. Here, we present a surface patterned with soft micrometric features inspired by the mushroom shape showing a nondestructive mechanical interlocking and thus attachment to fabrics. We provide a scalable experimental approach to prepare these surfaces and quantify the attachment strength with rheometric and video-based analysis. In these "probabilistic fasteners," we find that higher feature densities result in higher attachment force; however, the individual feature strength is higher on a low feature density surface. We interpret our results via a load-sharing principle common in fiber bundle models. Our work provides new handles for tuning the mechanical attachment properties of soft patterned surfaces that can be used in various applications including soft robotics.