Fibrillar Hydrogels Research Articles

Energy Landscape of the Sugar Conformation Controls the Sol-to-Gel Transition in Self-Assembled Bola Glycolipid Hydrogels

Self-assembled fibrillar network hydrogels and organogels are commonly obtained through a crystallization process as fibers upon being induced by external stimuli such as temperature or pH. The gel-to-sol-to-gel transition is generally readily reversible, and the rate of change of the stimulus determines the fiber homogeneity and eventual elastic properties of the gels. However, recent work shows that in some specific cases, fibrillation occurs for a given molecular conformation and the sol-to-gel transition depends on the relative energetic stability of one conformation over the other and not on the rate of change of the stimuli. We observe such a phenomenon on a class of bolaform glycolipids, sophorosides, similar to the well-known sophorolipid biosurfactants but composed of two symmetric sophorose units. A combination of oscillatory rheology, small-angle X-ray scattering (SAXS), cryogenic transmission electron microscopy, and in situ rheology coupled with SAXS using synchrotron radiation shows that below 14 °C, twisted nanofibers are the thermodynamic phase. Between 14 and about 33 °C, nanofibers coexist with micelles and a strong hydrogel forms, the sol-to-gel transition being readily reversible in this temperature range. However, above the annealing temperature of about 40 °C, the micelle morphology becomes kinetically trapped for hours, even upon cooling, whichever the rate, to 4 °C. A combination of solution and solid-state nuclear magnetic resonance spectroscopy studies suggests two different conformations of the 1″, 1′, and 2′ carbon stereocenters of sophorose, precisely at the β(1,2) glycosidic bond, for which several combinations of the dihedral angles are known to provide at least three energetic minima of comparable magnitude, with each corresponding to a given sophorose conformation.

Stiffening Matrix Induces Age-Mediated Microvascular Phenotype Through Increased Cell Contractility and Destabilization of Adherens Junctions.

Aging is a major risk factor in microvascular dysfunction and disease development, but the underlying mechanism remains largely unknown. As a result, age‐mediated changes in the mechanical properties of tissue collagen have gained interest as drivers of endothelial cell (EC) dysfunction. 3D culture models that mimic age‐mediated changes in the microvasculature can facilitate mechanistic understanding. A fibrillar hydrogel capable of changing its stiffness after forming microvascular networks is established. This hydrogel model is used to form vascular networks from induced pluripotent stem cells under soft conditions that mimic young tissue mechanics. Then matrix stiffness is gradually increased, thus exposing the vascular networks to the aging‐mimicry process in vitro. It is found that upon dynamic matrix stiffening, EC contractility is increased, resulting in the activation of focal adhesion kinase and subsequent dissociation of β‐catenin from VE‐Cadherin mediated adherens junctions, leading to the abruption of the vascular networks. Inhibiting cell contractility impedes the dissociation of β‐catenin, thereby preventing the deconstruction of adherens junctions, thus partially rescuing the age‐mediated vascular phenotype. The findings provide the first direct evidence of matrix's dynamic mechano‐changes in compromising microvasculature with aging and highlight the importance of hydrogel systems to study tissue‐level changes with aging in basic and translational studies.

Robust and highly adaptable high internal phase gel emulsions stabilized solely by a natural saponin hydrogelator glycyrrhizic acid.

Herein, we report a new class of high internal phase gel emulsions (gel-HIPEs) that are mechanically robust, adaptable, and processable. They can be synthesized facilely by using the natural food-grade saponin glycyrrhizic acid (GA) as the sole stabilizer, which is shown to be versatile for various oils. The structural properties of these HIPEs including appearance, viscoelasticity and processability are well controlled by simply changing the concentration of GA nanofibrils. When the GA nanofibril concentration exceeds 0.3 wt%, the unique gel-HIPEs can be produced through the formation of fibrillar hydrogel networks in the continuous phase. When the nanofibril concentration only increases to 5 wt%, it is surprising to see that these gel-HIPEs display an extremely high mechanical strength, and the storage moduli as well as the yield stress values can reach 408.5 kPa and 3340 Pa (or even more), respectively. We conjecture that such remarkable mechanical performance is mainly attributed to the highly viscoelastic GA nanofibrillar networks in the continuous phase of gel-HIPEs, which can actively trap the nanofibril-coated emulsion droplets and thus strengthen the gel matrix. Consequently, the robust gel-HIPEs can be used as a solid template to fabricate stable porous materials without the need for crosslinking of the continuous phase, and the open- and closed-cell foam microstructures are controlled by the nanofibril concentration. Furthermore, the nanofibril-based HIPEs are promising long-term delivery vehicles with controlled-release properties for lipophilic active cargoes, since the strong fibrillar networks at the droplet surfaces and in the continuous phase can effectively retard the active release.

Stable and biocompatible fibrillar hydrogels based on the self-crosslinking between collagen and oxidized chondroitin sulfate

Stable and biocompatible collagen-based hydrogels based on natural extracellular matrix (ECM) with a homogeneous microstructure are ideal scaffolds for tissue engineering; however, lack of structural stability and mechanical property have hindered their application. In this work, Chondroitin Sulfate (CS) was periodate-oxidized to prepare dialdehyde ECM-derived biopolymer with the weight average molecular weight of 3.7 × 104 g/moL to construct the stable collagen-based fibrillar hydrogels. The results of Fourier Transform Infrared (FTIR) analysis and hydroxylamine hydrochloride titration assay confirmed the presence of aldehyde groups in oxidized CS (CSox) with an oxidization degree of 30.67%. Gelation time test and scanning electron microscopy (SEM) observation showed that gel formation and fibrillogenesis process were accelerated by adding CSox, and all hydrogels exhibited a tight fibrillar structure. With an increase in CSox concentration from 0.125 to 2.000 mg/mL, the crosslinking degree increased from 24.95% to 35.05% due to the formation of covalent bonds between the ε-amino groups of collagen and aldehyde of CSox. The mechanical strength of collagen/CSox hydrogels reached a maximum value of 11.79 KPa, which was more than triple that of pure collagen hydrogel (3.58 KPa) at 0.125 mg/mL CSox. The swelling ratio, enzymatic resistance, and thermal stability were also improved with the increasing concentration of CSox. When the CSox concentration reached 0.500 mg/mL, the collagen-based hydrogels exhibited the highest thermal denaturation temperature, which was approximately 7.5 °C higher than that of the pure collagen hydrogel. Moreover, the results of the CCK-8 assay and LIVE/DEAD staining demonstrated that all the collagen/CSox hydrogels accelerated cell proliferation and showed acceptable biocompatibility. This work broadens the avenue for the design of natural collagen-based hydrogels for tissue engineering.

Poroviscoelasticity and compression-softening of agarose hydrogels

Agarose hydrogels are poroviscoelastic materials that exhibit a waterlogged-crosslinked microstructure. Despite an extensive use in biotechnologies and numerous studies of the elastic properties of agarose gels, little is known about the compressible behavior and the microstructural changes of such fibrillar hydrogels under compression. The present work investigates the mechanical response of centimeter-sized pre-molded agarose cylinders when applying a compressive strain ramp over an extended range of loading speed and polymer concentration. One of the original contributions is the simultaneous monitoring of the changes in the hydrogel volume to determine the Poisson’s ratio through a spatiotemporal method. The linear poroelastic response of agarose hydrogels shows a compressible behavior at strain rates less than 0.7 % s−1. The critical compressive strain of a few percent at the onset of the non-linear regime and the always positive Poisson’s ratio decrease when applying a slow compressive ramp. The mechanical response in the linear regime is typical of a deformation mode either dominated by the bending of semiflexible strands (enthalpic regime) or by the stretching of the network (entropic regime) at higher agarose concentration. Cyclic linear shear deformations superimposed to a compressive strain from 0.5 up to 40% further give evidence of a compression-softening of the network causing the transition to the non-linear regime without dependence upon the network topology and connectivity. Finally, the buckling-induced aging of the network under a weak compression and the poroviscoelasticity of the hydrogel are shown to impact the relaxation of the normal stress and the equilibrium stress.

Multiphase Assembly of Small Molecule Microcrystalline Peptide Hydrogel Allows Immunomodulatory Combination Therapy for Long-Term Heart Transplant Survival.

Combination therapies that target multiple pathways involved in immune rejection of transplants hold promise for patients in need of restorative surgery. Herein, a noninteracting multiphase molecular assembly approach is developed to crystallize tofacitinib, a potent JAK1/3 inhibitor, within a shear-thinning self-assembled fibrillar peptide hydrogel network. The resulting microcrystalline tofacitinib hydrogel (MTH) can be syringe-injected directly to the grafting site during surgery to locally deliver the small molecule. The rate of drug delivered from MTH is largely controlled by the dissolution of the encapsulated microcrystals. A single application of MTH, in combination with systemically delivered CTLA4-Ig, a co-stimulation inhibitor, affords significant graft survival in mice receiving heterotopic heart transplants. Locoregional studies indicate that the local delivery of tofacitinib at the graft site enabled by MTH is required for the observed enhanced graft survival.

Marangoni flows drive the alignment of fibrillar cell-laden hydrogels.

When a sessile droplet containing a solute in a volatile solvent evaporates, flow in the droplet can transport and assemble solute particles into complex patterns. Transport in evaporating sessile droplets has largely been examined in solvents that undergo complete evaporation. Here, we demonstrate that flow in evaporating aqueous sessile droplets containing type I collagen-a self-assembling polymer-can be harnessed to engineer hydrated networks of aligned collagen fibers. We find that Marangoni flows direct collagen fiber assembly over millimeter-scale areas in a manner that depends on the rate of self-assembly, the relative humidity of the surrounding environment, and the geometry of the droplet. Skeletal muscle cells that are incorporated into and cultured within these evaporating droplets collectively orient and subsequently differentiate into myotubes in response to aligned networks of collagen. Our findings demonstrate a simple, tunable, and high-throughput approach to engineer aligned fibrillar hydrogels and cell-laden biomimetic materials.

Dense fibrillar collagen-based hydrogels as functional osteoid-mimicking scaffolds

ABSTRACT There is an increasing need to generate novel materials for the treatment and augmentation of bone defects, affecting millions of people worldwide. Fibrillar type I collagen is the most abundant tissue matrix protein in bone, providing its key native scaffolding material. However, while in vitro reconstituted collagen hydrogels of physically entangled, nano-fibred meshes, have long served as three-dimensional cultures, their highly-hydrated nature impacts their physiological relevance. In an effort to create biomimetic collagen gels, approaches have been undertaken to generate osteoid-like environments with increased collagen concentrations, controlled fibrillar orientation, defined micro-architectures, and tailored mechanical properties. This review describes the state-of-the-art on collagen densification techniques, exploring their advantages, limitations and future perspectives for applications as bone grafts. Ultimately, by successfully mimicking the organic milieu of bone through acellular or cell-mediated mineralisation of the designed osteoid-like structure, functional collagen scaffolds with potential applications in bone tissue engineering can be realised. Abbreviations: 3D: three-dimensional; BG: bioactive glass; CFD: collagen fibrillar density; CHA: carbonated-hydroxyapatite; Col1: Type I collagen; ECM: extracellular matrix; GAE: gel aspiration-ejection; HHC: highly hydrated collagen; MSC: mesenchymal stem cell; NCPs: non-collagenous proteins; PC: plastic compression; PILP: polymer-induced liquid precursor; SBF: simulated body fluid

Integrated Biophysical Characterization of Fibrillar Collagen-Based Hydrogels.

This paper describes an experimental characterization scheme of the biophysical properties of reconstituted hydrogel matrices based on indentation testing, quantification of transport via microfluidics, and confocal reflectance microscopy analysis. While methods for characterizing hydrogels exist and are widely used, they often do not measure diffusive and convective transport concurrently, determine the relationship between microstructure and transport properties, and decouple matrix mechanics and transport properties. Our integrated approach enabled independent and quantitative measurements of the structural, mechanical, and transport properties of hydrogels in a single study. We used fibrillar type I collagen as the base matrix and investigated the effects of two different matrix modifications: (1) cross-linking with human recombinant tissue transglutaminase II (hrTGII) and (2) supplementation with the nonfibrillar matrix constituent hyaluronic acid (HA). hrTGII modified the matrix structure and transport but not mechanical parameters. Furthermore, changes in the matrix structure due to hrTGII were seen to be dependent on the concentration of collagen. In contrast, supplementation of HA at different collagen concentrations altered the matrix microstructure and mechanical indentation behavior but not transport parameters. These experimental observations reveal the important relationship between extracellular matrix (ECM) composition and biophysical properties. The integrated techniques are versatile, robust, and accessible; and as matrix-cell interactions are instrumental for many biological processes, the methods and findings described here should be broadly applicable for characterizing hydrogel materials used for three-dimensional (3-D) tissue-engineered culture models.

Engineering rhizobacterial community resilience with mannose nanofibril hydrogels towards maintaining grain production under drying climate stress

Ongoing impacts of climatic change especially moisture stress remain a global challenge to agricultural production and food security. Such abiotic effects directly influence soil microbial communities. Previously we demonstrated that polymeric hydrogels, able to provide specific interactions with soil microbial communities, enhance the dynamics and selectivity of ingress and colonisation of plant beneficial bacteria at soil microcosm scales. We now show that small quantities of osmotic hydrogels containing mannose nanofibrils specifically situated in close proximity to developing root zones provide enhanced microbial ingress, colonisation and continuous wetting pathways when contacting developing wheat root systems. These effectively create extensions to the natural rhizosphere sustaining significant beneficial rhizobacterial communities when under moisture stress. Sequencing studies, on wheat production soils undergoing a season of low average rainfall clearly showed microbial abundance increases and taxa selectivity in the rhizosphere-hydrogel regions compared to controls. Here wheat yields increased by about 20% with hydrogel addition compared to controls, which represented a 15% increase in the overall average yield in the region. This represents the first reported demonstration that developing rhizospheres may be readily engineered to consistently select their own distinct microbiome as nodes of functional microbial abundance significantly benefiting grain yield during abiotic stress. Thereby suggesting new opportunities to maintain grain yields during periods of drying climate via these fibrillar hydrogels.

Food-Grade Emulsions and Emulsion Gels Prepared by Soy Protein-Pectin Complex Nanoparticles and Glycyrrhizic Acid Nanofibrils.

Glycyrrhizic acid (GA) and GA nanofibrils (GN) have been shown to be efficient natural emulsifiers for formation and stabilization of food emulsions. In this work, the emulsion properties of GN in the presence of soy protein isolate-pectin complex nanoparticles (SPNPs) were studied to understand the impact of the nanofibril-nanoparticle interactions on emulsion stabilization. In the presence of low GN concentrations (0.1-0.5 wt %), the synergy in reducing the interfacial tension was observed due to SPNPs-GN complexation in the bulk and at the interface by hydrogen bonding, endowing the prepared emulsions with an overall smaller droplet size. However, obvious flocculation and clustering of oil droplets occurred in these emulsions (especially at 0.25 and 0.5 wt % GN), which are probably induced by a depletion mechanism. At high GN concentrations (1-2 wt %), due to the preferential adsorption, the GN mainly dominated the interface and the subsequent formation and properties of emulsions. Accordingly, the self-standing emulsion gels were obtained, showing a small droplet size with d32 of about 1.0-1.5 μm, homogeneous appearance and microstructure, and encouraging rheological properties including high gel strength, shear sensitivity, and good thixotropic recovery. This is mainly attributed to the formation of a fibrillar hydrogel network in the continuous phase as well as around the droplet surfaces.

Electrostatically Driven Guanidinium Interaction Domains that Control Hydrogel-Mediated Protein Delivery In Vivo.

Protein biologics are an important class of drugs, but the necessity for frequent parenteral administration is a major limitation. Drug-delivery materials offer a potential solution, but protein-material adsorption can cause denaturation, which reduces their effectiveness. Here, we describe a new protein delivery platform that limits direct contact between globular protein domains and material matrix, yet from a single subcutaneous administration can be tuned for long-term drug release. The strategy utilizes complementary electrostatic interactions made between a suite of designed interaction domains (IDs), installed onto the terminus of a protein of interest, and a negatively charged self-assembled fibrillar hydrogel. These intermolecular interactions can be easily modulated by choice of ID to control material interaction and desorption energies, which allows regulation of protein release kinetics to fit desired release profiles. Molecular dynamics studies provided a molecular-level understanding of the mechanisms that govern release and identified optimal binding zones on the gel fibrils that facilitate strong ID–material interactions, which are crucial for sustained release of protein. This delivery platform can be easily loaded with cargo, is shear-thin syringe implantable, provides improved protein stability, is capable of a diverse range of in vitro release rates, and most importantly, can accomplish long-term control over in vivo protein delivery.

New Formulation with Marine Algae from Black Sea

Seaweed is a natural treasure that can be intensely evaluated for therapeutic purposes. During the past years, it became obvious that the ecosystem presents a marine algae excedent, which should be utilized in one way or another. Marine algae have been intensely studied. The superior exploitation of the marine biomass represents a highly important resource for the pharmaceutical industry, supplying raw material for the extraction of bioactive substances (vitamins, sterols, and aminoacids) and various other substances, the purity of which is strongly connected to the state of the marine ecosystem. In present work the extracts from marine algae are incorporated in type I non-denatured fibrillar collagen matrixes for obtain new pharmaceutical product. In order to obtain therapeutic effects at nanostructure level, it is important to know the rheological characteristics of the relevant mixtures of collagen gels and extracts from marine algae selected for use. In this survey we have studied mixtures made of non-denatured fibrillar collagen hydro-gels where different concentrations of marine algae have been incorporated.

A Natural Supramolecular Saponin Hydrogelator for Creation of Ultrastable and Thermostimulable Food‐Grade Foams

AbstractA new class of food‐grade foams that are ultrastable, thermostimulable, and processable can be created simply by using the naturally occurring saponin glycyrrhizic acid (GA) as the sole stabilizer. The creation of this “superfoam” is based on the spatially controllable self‐assembly of supramolecular GA nanofibril hydrogelators at the air–water interface and in the continuous phase. The rapid adsorption of GA nanofibrils at the bubble surface, forming a multilayer interfacial network, combined with the formation of viscoelastic fibrillar hydrogel networks in the continuous phase, enables the foams having ultrastability over months or years without the water drainage induced phase separation, which have been evidenced using small angle X‐ray scattering and microscopy techniques. Such ultrastable foams can be rapidly destabilized on demand by heating, which induces the melting of the fibrillar networks. These thermoresponsive foams can be reversibly switched between stable and unstable by simply changing the temperature, based on the reversible gel–sol phase transition of the supramolecular hydrogel inside the foam. This is the first finding of a natural edible surfactant system that foams very well and can be used solely to make advanced foams with the qualities of simplicity, ultrastability, stimulability, and processability, which make them viable for many sustainable applications.

Stimuli Responsive, Self-Sustainable, and Self-Healable Functionalized Hydrogel with Dual Gelation, Load-Bearing, and Dye-Absorbing Properties.

The motivation for designing low-molecular-weight gelators with self-healing characteristics originates from elegant examples in biology such as vines of the genus Aristolochia whose internal secondary growth exhibits rapid self-healing in their stems. In the present work, we had explored the stimuli-responsive dual gelation characteristics for the ester-functionalized surfactant (4-(2-(hexadecyloxy)-2-oxoethyl)-4-methylmorpholin-4-ium bromide, C16EMorphBr) in aqueous medium at 7.20% (w/v) critical gel concentration and pH 7.4. The hydrogel provides an excellent platform to study dynamic phase behavior within a supramolecular network as it exhibits transformation from a fibrillar opaque hydrogel to a transparent hydrogel upon heating. Molecular interactions, arrangement within the supramolecular framework, and mechanical properties of the hydrogels were characterized using Fourier transform infrared, small-angle neutron scattering, rheological analysis, and tensile strength and cyclic loading-unloading tests. The fibrillar opaque gel has been characterized for its morphology using scanning electron microscopy, field emission scanning electron microscopy, transmission electron microscopy, and atomic force microscopy. The self-sustained, self-healable porous fibrillar opaque xerogel was further explored for selectively absorbing anionic dyes and for its load-bearing characteristics. We conclude a perspective on designing a new-age gelator that can open entirely new avenues in environmental protection and wearable "smart" devices.

Design and applications of man-made biomimetic fibrillar hydrogels

Many extracellular matrices (ECMs) have a filamentous architecture, which influences cell growth and phenotype and imparts tissues with specific properties. Man-made fibrillar hydrogels can function as biomimetic materials to reproduce the filamentous nature and properties of ECMs and to serve as scaffolds for 3D cell culture and tissue engineering. Different types of synthetic nanofibrillar hydrogels have been developed, with diverse mechanisms of assembly and a variety of physical properties and applications. In this Review, we explore the design and properties of biomimetic man-made nanofibrillar hydrogels. We discuss the assembly of peptides, block copolymer worm-like micelles and filamentous nanoparticles into fibrillar hydrogels and investigate the relationship between structure and physical as well as biochemical properties. Potential applications for 3D cell culture and tissue engineering are examined, and the properties and structure of natural and man-made fibrillar hydrogels are compared. Finally, we critically assess current challenges and future directions of the field. Man-made fibrillar hydrogels mimic the structure of filamentous extracellular matrices and can be used as biomaterials for 3D cell culture and tissue engineering. In this Review, the authors discuss the design and properties of fibrillar hydrogels and explore different building blocks, assembly mechanisms, properties and applications.

Cu/Zn-superoxide dismutase forms fibrillar hydrogels in a pH-dependent manner via a water-rich extended intermediate state.

Under certain conditions, amyloid-like fibrils can develop into three-dimensional networks and form hydrogels by a self-assembly process. When Cu/Zn superoxide dismutase (SOD1), an anti-oxidative enzyme, undergoes misfolding, fibrillar aggregates are formed, which are a hallmark of a certain form of familial amyotrophic lateral sclerosis (ALS). However, the issue of whether SOD1 fibrils can be assembled into hydrogels remains to be tested. Here, we show that the SOD1 polypeptides undergo hydrogelation accompanied by the formation of thioflavin T-positive fibrils at pH 3.0 and 4.0, but not at pH 5.0 where precipitates are formed. The results of viscoelastic analyses indicate that the properties of SOD1 hydrogels (2%) were similar to and slightly more fragile than a 0.25% agarose gel. In addition, monitoring by a quartz crystal microbalance with admittance analysis showed that the denaturing of immobilized SOD1 on a sensor under the hydrogelation conditions at pH 3.0 and 4.0 resulted in an increase in the effective acoustic thickness from ~3.3 nm (a folded rigid form) to ~50 and ~100 nm (an extended water-rich state), respectively. In contrast, when SOD1 was denatured under the same conditions at pH 5.0, a compact water-poor state with an effective acoustic thickness of ~10 nm was formed. The addition of physiological concentrations of NaCl to the pH 4.0 sample induced a further extension of the SOD1 with larger amounts of water molecules (with an effective acoustic thickness of ~200 nm) but suppressed hydrogel formation. These results suggest that different denatured intermediate states of the protein before self-assembly play a major role in determining the characteristics of the resulting aggregates and that a conformational change to a suitable level of extended water-rich intermediate state before and/or during intermolecular assembling is required for fibrillation and hydrogelation in the case of globular proteins.

Construction and evaluation of fibrillar composite hydrogel of collagen/konjac glucomannan for potential biomedical applications.

Konjac glucomannan (KGM) is recognized as a safe material for its health-promoting benefits and thus widely used in various fields including pharmaceutical industry. In recent decades, the combination of collagen and KGM attracts more attentions for biomedical purpose, especially the hybrid films of collagen–KGM or collagen–KGM–polysaccharide. In this study, to further and deeply develop the intrinsic values of both collagen and KGM as biomaterials, a novel kind of composite hydrogel comprising collagen and KGM at a certain ratio was fabricated under mild conditions via fibrillogenesis process of the aqueous blends of collagen and KGM that experienced deacetylation simultaneously. The chemical composition, microcosmic architectures, swelling behavior, biodegradation and dynamic mechanic properties of such resulted composite hydrogels were systematically investigated. Biologic experiments, including cell culture in vitro and hypodermic implantation in vivo, were also conducted on these collagen/KGM composite hydrogels to evaluate their biologic performances. The relevant results prove that, based on collagen self-assembly behavior, this synthesis strategy is efficient to construct a composite hydrogel of collagen/KGM with improved mechanical properties, biodegradability, excellent biocompatibility and bioactivity, which are promising for potential biomedical applications such as tissue engineering and regenerative medicine.

The Effect of Lipidation on the Self-Assembly of the Gut-Derived Peptide Hormone PYY3-36.

Lipidation is a powerful strategy to improve the stability in vivo of peptide drugs. Attachment of a lipid chain to a hydrophilic peptide leads to amphiphilicity and the potential for surfactant-like self-assembly. Here, the self-assembly and conformation of three lipidated derivatives of the gastrointestinal peptide hormone PYY3-36 is examined using a comprehensive range of spectroscopic, scattering, and electron microscopy methods and compared to those of the parent PYY3-36 peptide. The peptides are lipidated at Ser(11), Arg(17), or Arg(23) in the peptide; the former is within the β-turn domain (based on the published solution NMR structure), and the latter two are both within the α-helical domain. We show that it is possible to access a remarkable diversity of nanostructures ranging from micelles to nanotapes and fibrillar hydrogels by control of assembly conditions (concentration, pH, and temperature). All of the lipopeptides self-assemble above a critical aggregation concentration (cac), determined through pyrene fluorescence probe measurements, and they all have predominantly α-helical secondary structure at their native pH. The pH and temperature dependence of the α-helical conformation were probed via circular dichroism spectroscopy experiments. Lipidation was found to provide enhanced stability against changes in temperature and pH. The self-assembled structures were investigated using small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM). Distinct differences in nanostructure were observed for lipidated and unlipidated peptides, also depending on the position of lipidation. Remarkably, micelles containing lipopeptides with α-helical peptide conformation were observed. Gelation was observed at higher concentrations in certain pH intervals for the lipidated peptides, but not for unlipidated PYY3-36. Thus, lipidation, in addition to enhancing stability against pH and temperature variation, also provides a route to prepare PYY peptide hydrogels. These findings provide important insights into the control of PYY3-36 conformation and aggregation by lipidation, relevant to the development of future therapeutics based on this peptide hormone, for example, in treatments for obesity.

Nuclear-Import Receptors Reverse Aberrant Phase Transitions of RNA-Binding Proteins with Prion-like Domains

RNA-binding proteins (RBPs) with prion-like domains (PrLDs) phase transition to functional liquids, which can mature into aberrant hydrogels composed of pathological fibrils that underpin fatal neurodegenerative disorders. Several nuclear RBPs with PrLDs, including TDP-43, FUS, hnRNPA1, and hnRNPA2, mislocalize to cytoplasmic inclusions in neurodegenerative disorders, and mutations in their PrLDs can accelerate fibrillization and cause disease. Here, weestablish that nuclear-import receptors (NIRs) specifically chaperone and potently disaggregate wild-type and disease-linked RBPs bearing a NLS. Karyopherin-β2 (also called Transportin-1) engages PY-NLSs to inhibit and reverse FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2 fibrillization, whereas Importin-α plus Karyopherin-β1 prevent and reverse TDP-43 fibrillization. Remarkably, Karyopherin-β2 dissolves phase-separated liquids and aberrant fibrillar hydrogels formed by FUS and hnRNPA1. Invivo, Karyopherin-β2 prevents RBPs with PY-NLSs accumulating in stress granules, restores nuclear RBP localization and function, and rescues degeneration caused by disease-linked FUS and hnRNPA2. Thus, NIRs therapeutically restore RBP homeostasis and mitigate neurodegeneration.