Biomacromolecules

Self-assembled peptide-based nanostructures have diverse applications in the pharmaceutical and materials fields, but accurately predicting their self-assembly behavior without time-intensive organic synthesis and characterization remains a significant challenge. Here, we assess the effectiveness of AlphaFold3 (AF3), a deep learning model for protein structure prediction, in modeling peptide-based nanostructures and the interactions driving supramolecular self-assembly. We designed amphiphilic peptides composed of alternating hydrophobic residues (valine, leucine, isoleucine, phenylalanine) and hydrophilic residues (glutamic acid), varying both sequence length and residue order. Using AF3s multimer mode, we modeled assemblies with copy numbers ranging from 10 to 1000, generating diverse morphologies such as micelles and nanotubes. We qualitatively analyzed hydrophobic regions, secondary structures, and intermolecular interactions, while also calculating radii of gyration, packing scores, and aspect ratios using PyRosetta. Our results indicate that AF3 predicts morphologies consistent with hydrophobic driving forces and steric constraints. Increased hydrophobicity correlates with smaller radii of gyration, while higher copy numbers correspond to smaller aspect ratios (more compact structures). Longer hydrophobic segments lead to disordered structures, whereas longer hydrophilic segments promote organization. While AF3 captures systemic trends consistent with biophysical principles, comparisons to literature reveal discrepancies driven by charge effects and secondary structure bias, including an overemphasis on helical propensity (e.g., alanine-rich sequences) and sensitivity to terminal charge repulsion. Additionally, since AF3 is predisposed to predict a single assembled entity rather than higher-order assemblies such as multiple micelles or fibers, finding the optimal copy number for the best prediction requires system-specific iteration. These limitations highlight the need for complementary approaches with controlled chemical potential and environmental conditions, though qualitative agreement with experimental trends in morphology and compactness supports AF3s utility for initial structure generation. Our findings highlight AF3s potential as a user-friendly design tool for structure generation in peptide design, aiding the efficient development of functional self-assembled peptide nanomaterials.

Solid-phase peptide synthesis (SPPS) remains the dominant technique for peptide production. However, its reliance on hazardous organic solvents such as N, N-dimethylformamide (DMF) and dichloromethane (DCM) results in an adverse environmental burden. One potential approach is replacing these organic solvents with water to reduce the hazardous solvent consumption and improve the environmental footprint of peptide production. This has led to the emergence of aqueous solid-phase peptide synthesis (ASPPS) approaches. Although successful, these approaches require specialized hydrophilic resins or modified building blocks, limiting their industrial applicability and scalability. Moreover, conventional hydrophobic polystyrene supports, remain the most widely used solid supports in industrial SPPS due to their high loading capacity, mechanical robustness, and low cost. These resins are generally considered incompatible with aqueous conditions. Here, we demonstrate that industrially relevant 2-chlorotrityl chloride (CTC) polystyrene resin can support efficient peptide coupling under fully aqueous conditions by integrating a precipitate-free 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC{middle dot}HCl) and Oxyma activation system with a synergistic thermal-acoustic strategy. We posit that heating combined with ultrasonic irradiation likely promotes transient relaxation of the polystyrene matrix and enhances water penetration. This facilitates the diffusion of activated amino acid esters onto the hydrophobic resin required for coupling. The robustness of this aqueous methodology was validated through the synthesis of nine structurally diverse peptide sequences, including aromatic hydrogel-forming peptides, opioid peptides derived from enkephalins, toxin-inspired sequences, and a lipid-interacting fragment of -synuclein. Analytical characterization by HPLC and MALDI-TOF mass spectrometry confirmed successful peptide assembly with high crude purity. We anticipate that this thermal-acoustic aqueous SPPS strategy provides a scalable and accessible pathway toward sustainable peptide manufacturing on classical hydrophobic supports with aqueous chemistry.

The widespread use of synthetic food dyes, such as Acid Yellow 23 (AY 23), in the food, cosmetics, and pharmaceutical industries raises questions about their potential effects on biological systems and public health. The concentration-dependent interaction between AY 23 and bovine serum albumin (BSA), a crucial model protein for understanding pharmacokinetics and protein-ligand behaviour, was examined in this study. We demonstrate that, under physiological conditions, increasing dye concentrations from 50 M to 200 M results in notable conformational changes, increased surface hydrophobicity, and protein aggregation using a multimodal biophysical approach that includes fluorescence spectroscopy. Direct visualisation verified these structural changes and aggregate formation, whereas hemolytic assay confirmed the high hemolytic nature of AY 23-induced fibrils. Additionally, this study provides a mechanistic basis for the toxicological effects of AY 23, underscoring the implications of food dyes for public health.

Control over network dynamics at different length scales is a feature of natural materials challenging to replicate in synthetic hydrogels. Hydrogel viscoelasticity is commonly controlled by tuning the kinetics of reversible crosslinks; however, this strategy inherently links the resulting macroscale and nanoscale dynamics of the individual network components. Taking inspiration from biological materials that feature lipids as structural elements, we introduce Lipid Network Crosslinked (LINC) hydrogels that exploit the mobility of individual lipids within self-assembled liposomes as covalent, network-crosslinking points. These mobile, covalent crosslinks increase hydrogel stress relaxation rates over 20-fold compared to polymer-only hydrogels with equivalent crosslinking chemistries and stiffnesses. We demonstrate that liposome design parameters, including degree of surface functionalization and tail saturation, provide a means to independently control the macroscale storage moduli and stress relaxation behavior. Finally, as an application where control over network dynamics at different length scales is critical, we placed cell-adhesive ligands onto more mobile or less mobile network elements. Human neural progenitor cells cultured within LINC hydrogels of identical macroscale viscoelasticity significantly altered their phenotype in response to nanoscale ligand dynamics. These results establish LINC hydrogels as biomimetic materials that leverage nanoscale lipid mobility within a macroscale polymeric network to control dynamics at multiple length scales.

Hydrogels are widely used as three-dimensional cell culture systems to understand the impact of cellular mechanotransduction for tissue engineering applications. Photoinitiated thiol-ene click chemistry is a commonly utilized hydrogel crosslinking mechanism that provides spatial and temporal control over hydrogel network formation and resulting mesh size and compressive properties. Despite historically documented efficiency as step-growth reactions, these reactions do not always proceed as predicted. To understand the impact of cell confinement and microenvironmental mechanics on cellular function, thiol-ene network formation must be thoroughly characterized. To this end, the objective of this work was to investigate the crosslinking dynamics to determine hydrogel network formation as assessed via mesh size and mechanical properties using a pentenoate-functionalized hyaluronic acid thiol-ene reaction. Hydrogel parameters including polymer concentration and thiol:-ene crosslinker molar ratio were modulated (4, 6, or 8 polymer weight percent and 0.15:1, 0.5:1, or 1:1 molar ratio of thiol groups to reactive -ene groups) to tune network properties including shear storage modulus and relative mesh size. Molecular Dynamics (MD) simulations were used to simulate the thiol-ene crosslinking reaction and establish a method for predicting thiol-ene reaction efficiency. Lastly, the feasibility of this hydrogel system for in vitro modeling was confirmed via assessment of metabolic activity of encapsulated primary human meniscal cells.

The hierarchical organization of cellulose microfibrils and their intimate interactions with hemicelluloses such as xylan underpin the exceptional mechanical performance of plant cell walls. However, translating these biological design principles into sustainable nanocellulosic materials remains limited by conventional cellulose nanofibril production routes, which rely on harsh chemical treatments that disrupt the native cellulose-hemicellulose architecture. Here, we present an optimized isolation strategy for holocellulose nanofibrils (hCNFs) that preserves native cellulose structure, xylan substitution and conformation, and cellulose-xylan interactions. Using wild-type Arabidopsis thaliana, a xylan glucuronidation-deficient gux1/2 mutant, and Brassica napus straw as model systems, we systematically elucidate how xylan content and substitution pattern govern nanofibril isolation, interfacial interactions, and macroscopic properties. Two-dimensional 13C magic-angle spinning NMR demonstrates retention of native cellulose glucosyl environments, the presence of two-fold and three-fold helical xylan conformations, and cellulose-associated two-fold helical xylan. Cryogenic transmission electron microscopy reveals fibril widths of [~]3 nm, consistent with elementary cellulose I{beta} microfibrils. We show that xylan glucuronidation regulates colloidal stability, hydration behavior, and interfibrillar cohesion, whereas xylan content controls nanofibrillation efficiency. These multiscale structural features translate directly into moisture sorption, thermal behavior, and mechanical performance. Notably, Brassica napus hCNF films exhibit exceptional strength and extensibility, surpassing many chemically modified CNF systems. This work demonstrates that preserving the native cellulose-hemicellulose architecture enables high-performance, sustainable nanocellulosic materials without chemical reconstruction.

Controlled release systems for subcutaneous peptide delivery often exhibit a pronounced initial burst release followed by inadequate maintenance of therapeutic exposure, limiting depot lifetime and increasing pharmacokinetic variability. Here, we engineer a dynamic, injectable hydrogel depot technology for months-long delivery of lipidated peptides. Using semaglutide as a model, we establish a modular formulation framework integrating: (i) formulation-driven tuning of depot mechanics to control release kinetics, (ii) cargo complexation strategies leveraging hydrophobic and multivalent ion-mediated interactions, and (iii) oxidative stabilization through sacrificial antioxidant excipients. We evaluated depot performance by rheology, in vitro cargo release, and in vivo pharmacokinetic and pharmacodynamic studies in rodents. Optimized formulations sustained semaglutide exposure for over six weeks from a single administration with two-fold reduction in peak-to-trough exposure and comparable total bioavailability relative to daily dosing, resulting in improved glucose control, weight regulation, and preservation of pancreatic islet content. These results suggest potential for quarterly dosing in humans. Together, this work establishes integrated and generalizable structure-property-performance relationships that account for cargo-matrix and cargo-excipient interactions across burst, diffusion, and erosion regimes to inform a practical formulation framework for engineering long-acting depots for sustained peptide delivery.

Recent advances in the discovery, characterisation, and engineering of urethanases provide new opportunities for the sustainable biocatalytic degradation of polyurethane waste. A mechanistic understanding of enzyme-plastic interactions is essential for structure-based engineering to enhance urethanase activity. However, the extremely complex and hydrophobic nature of polyurethane makes it challenging to elucidate the structural basis of enzyme-plastic interactions. Here, we used a fragment-based approach to characterise the active sites of two novel urethanases with different catalytic scaffolds, employing both a crystallographic fragment-screening (FASE) campaign and soluble fragments of plastic-like analogues that mimic the substrate, transition state, or product. FASE identified new substrate-binding subpockets while interactions of plastic mimetics in the active site provided a mechanistic understanding of the recognition and binding of polyurethane fragments by these subpockets. These results highlight a diversity of binding modes among urethanases toward different polyurethane fragments.

Bacterial microcompartments (BMCs) are a diverse and widespread class of protein-based organelle consisting of a semi-permeable protein shell encapsulating an enzymatic core. Along with their native assembly pathway, isolated BMC shell proteins have been shown to assemble into alternative superstructures such as flat sheets and nanotubes. The self-assembly and modularity of BMC shell proteins make them of great interest as modular platforms for applications involving scaffolding, immobilization and compartmentalization. While the assembly of BMC shell proteins into higher-order structures has been well-studied, the design of controllable and modular cargo loading is underdeveloped in comparison. Recently, we reported the pH-controlled assembly of CcmK2 - the major hexameric shell protein of the {beta}-carboxysome BMC - into monodisperse mesh-like microscale particles. Here, we develop a suite of encapsulation strategies for stochastic or targeted loading of various cargos, as well as the direct conjugation of cargo to CcmK2 particles. Our systematic analysis demonstrates that cargo loading and particle assembly can be modulated by the choice of recruitment strategy and the order of cargo introduction. Our findings also reveal a cooperative cargo loading mechanism during assembly that influences particle sizing and apparent morphology. Our study serves as a blueprint for the rational design of tunable cargo loading into engineered BMC-derived microcompartment systems for diverse biotechnological applications.

With the push towards accessible benchtop models to capture biological events, many researchers are reaching for hydrogel platforms for 3D tissue engineering ex vivo. Recapitulating the dynamic mechanical environment cells experience in vivo requires dynamic hydrogel scaffolds whose mechanical properties can be reprogrammed with spatiotemporal precision. Here we describe a chemically simple hydrogel platform that undergoes visible-light photosoftening via a ruthenium-based photocleavable crosslinker, leveraging tetrazine-norbornene inverse electron demand Diels Alder (iEDDA) click chemistry between RuTetrazine crosslinker and norbornene-modified hyaluronic acid (NorHA). Nitrogen gas evolved during this reaction is repurposed as an intrinsic porogen, nucleating macropores (55-175 {micro}m) directly during gelation. Initial stiffness (1.5-10 kPa) and softening extent (from 50%-100% drop in storage modulus) are independently tunable through polymer and crosslinker composition. We have found RuTetrazine to be non-mutagenic and non-toxic (>80% live cell populations) once network-bound (IC50 = 0.27 mM). In a cell-instructive network co-crosslinked with an MMP-RGD-bearing peptide, human mesenchymal stromal cells (hMSCs) photosoftened in situ (2.27[-&gt;]0.54 kPa, [~]76%) spread approximately six-fold relative to stiff controls ([~]6,500 vs. [~]1,100 {micro}m2, p < 0.0001). This work demonstrates a synthetically accessible photocleavable crosslinker and a simple, macroporous hydrogel for modulating dynamic mechanical cues in three dimensions.

Poly(ethylene terephthalate) (PET), a ubiquitous polyester used in packaging and textiles, persists in the environment due to its high crystallinity and stability, contributing substantially to global plastic pollution. Enzymatic depolymerization by PET hydrolase (PETase) offers a chemically precise and environmentally sustainable route to convert PET into its monomeric building blocks, enabling recycling. However, practical implementation remains hindered by the rigid, crystalline architecture of PET, which restricts enzyme access and necessitates energy-intensive pretreatment to enable efficient depolymerization. In addition, most PETases achieve only partial depolymerization with low terephthalic acid (TPA) yields and accumulate inhibitory intermediates, while limited thermostability and slow surface kinetics further restrict efficiency. To overcome these barriers, we report VenusPETase, an engineered variant of KbPETase designed using PET-Flow, a state-of-the-art computational framework for PETase engineering. Compared with KbPETase, VenusPETase exhibits a 3.4-fold increase in hydrolytic activity, up to a 27-fold improvement after heat treatment of protein, and a 12 {degrees}C enhancement in thermostability. VenusPETase exhibits rapid degradation across a wide crystallinity range (8%-50%) at 50 {degrees}C, effectively spanning the entire spectrum of commercial PET products. Moreover, VenusPETase outperformed nine high-performance PETases under their respective optimal conditions and degraded untreated PET substrates across 8%-50% crystallinity, producing TPA as over 95% of the released products with minimal intermediate accumulation. X-ray crystallography and molecular dynamics simulations suggest that dynamic modulation, elevated surface electrostatic potential enhance the interaction of VenusPETase with crystalline PET, thereby lowering the hydrolytic energy barrier and improving catalytic performance. We also demonstrate that untreated, postconsumer-PET from nine different products can all be degraded by VenusPETase. The recovered monomers can be directly repolymerized into virgin-quality PET, demonstrating a closed-loop enzymatic recycling process. In a 100 L bioreactor, VenusPETase completely depolymerizes post-consumer crystalline PET (28% crystallinity) within 24 h under 50 {degrees}C. These results establish VenusPETase as a robust biocatalyst that enables efficient, closed-loop recycling of crystalline PET under mild conditions.

Sustainable, biodegradable elastomers are needed to replace fossil-based alternatives and reduce the environmental impact of traditional vibration damping materials. We investigate agarose-based hydrogels as eco-friendly vibration absorbers, examining the combined effects of polymer concentration (1-7 wt%), relative humidity (55-98%), and mechanical pre-stress on their dynamic mechanical properties. Frequency-dependent viscoelastic and vibration transmissibility tests, supported by Gaussian Process Regression (GPR), reveal that increasing agarose concentration enhances the storage modulus (E') by over an order of magnitude, reaching[~] 5 MPa depending on humidity and applied prestress. Remarkably, the damping efficiency--characterised by the loss factor (tan(d))--exhibits a highly non-monotonic trend. Maximum energy dissipation is observed at intermediate network densities, with tan(d) up to 0.21 and a loss modulus of[~] 515 kPa at 5 w% and 75% relative humidity, comparable to synthetic elastomers. GPR analysis shows that prestress controls nonlinear stiffening and transmissibility resonance behavior, while shifting peak damping from 5 wt% to 1 wt% agarose as prestress increases. These findings underscore the mechanical tunability and sustainability of agarose hydrogels, providing potential design guidance for biodegradable vibration mitigation materials.

Agricultural mulch films improve crop productivity, but post-use recovery and recycling remain difficult because the films are thin, fragmented, dispersed across fields, and contaminated. Commercial PLA/PBAT blends are increasingly used as biodegradable mulch film materials, yet these films exhibit slow or incomplete degradation under environmental conditions. Here, we show that Kubu, a thermostable PETase from Kutzneria buriramensis, rapidly depolymerizes commercial PLA/PBAT mulch film without pretreatment at 60 {degrees}C, achieving 95% mass loss within 96 h and releasing terephthalate, adipate, and lactate, detected by LC-MS/MS and HPLC, as the major monomeric products of both PBAT and PLA components. GPC and SEM revealed extensive degradation at the polymer-water interface. DiffDock docking against Kubu, IsPETase, and TfCut indicates that the canonical W/F(Y) cleft of the PETase/Cutinase fold accommodates aliphatic and aromatic ester bonds with comparable geometry, suggesting that substrate promiscuity is an inherent property of the cleft architecture. Kubus distinctive contribution combines this permissive cleft with catalytic activity sufficient for near-complete blend depolymerization within 24 h. Integration with a socioeconomic analysis shows that complete enzymatic depolymerization could avoid social costs up to $4,087 per ton of mulch film. These findings establish a single-enzyme approach to end-of-life management of heterogeneous polyester blends.

Protein cages provide useful scaffolds for nanoscale engineering due to their highly ordered structures and in vivo self-assembly. These scaffolds are amendable to late-stage conjugation, enabling expansion in functionality. However, many conjugation techniques either lack site-selectivity, require unnatural amino acid incorporation, or have bulky recognition motifs to facilitate ligation reactions. Here, an asparaginyl endopeptidase (AEP) enzyme with ligase activity is employed for the highly efficient functionalization of virus-like particles (VLPs) from Salmonella Typhimurium bacteriophage P22. The capacity of this enzyme to conjugate peptides and proteins onto assembled P22 VLPs under mild reaction conditions, via a minimal extension to the P22 coat protein C-terminus, is demonstrated. We extend the reaction efficiency to facilitate a one-pot dual-functionalization reaction whereby two therapeutically relevant receptor targeting domains are conjugated to P22 VLPs in a single step. Finally, we demonstrate the potential for AEP-mediated bioconjugation to bestow P22 VLPs with receptor-binding functionality in vitro. This work demonstrates the efficacy of AEP ligases as bioconjugation tools for site-selective functionalization of large molecular assemblies like VLPs.

Silk proteins, including sericin and fibroin, are natural biopolymers with broad applications in tissue engineering where angiogenesis plays an essential role. However, the pro-angiogenic effects of silk proteins with varying molecular weights (MWs) remain poorly understood. Here, silk proteins with MW distributions at 40-180 kDa or less than 25 kDa were obtained through alkaline hydrolysis to evaluate their effects on angiogenesis. Structurally, reducing MW induced a conformational transition in silk proteins, accompanied by a striking morphological shift in sericin from nanofibers to nanoparticles. Functionally, high-MW sericin (SSH) suppressed, whereas low-MW sericin (SSL) and both high- and low-MW silk fibroin (SFH/SFL) directly promoted endothelial angiogenic activity. Transcriptomic analysis revealed that angiogenesis-related genes such as Id1 and Smad6/9 may underlie the angiostatic effects of SSH. Notably, both SSH and SSL enhanced angiogenesis indirectly via macrophages; however, SSH induced mixed M1/M2-like polarization, while SSL preferentially drove an M2-like phenotype. In a subcutaneous implantation model, SSH promoted angiogenesis but yielded vessels with weak integrity and increased fibrosis, whereas SSL enhanced angiogenesis with improved vascular maturity and reduced fibrotic response. These findings elucidate how the MWs of silk proteins shape angiogenic behavior and highlight the importance of MW tailoring for optimized tissue engineering applications.

Cell penetrating thyclotides (CPTs) are synthetic molecules that promote highly efficient cellular uptake and endosomal escape of bioactive peptides. While peptides are valuable as medicinal agents, their translation to therapies is often limited by their inability to cross cell membranes. CPTs have a unique combination of chiral tetrahydrofurans and polar sidechains within a molecular scaffold that can be optimized to efficiently deliver peptide cargo into cells. The cellular uptake and endosomal escape of two peptides with anticancer biological activities but low bioavailabilities were remarkably improved after conjugation to a CPT. Using CPTs to overcome barriers to cellular uptake represents a new direction for the intracellular delivery of bioactive molecules, and will accelerate drug development for new medical therapies.

Cell migration in three-dimensional (3D) environments is highly plastic and regulated by extracellular matrix (ECM) cues. Engineered biomaterials provide controllable platforms to investigate how specific matrix signals regulate cell behavior in 3D, yet how defined biochemical signals control migration modes remain unclear. Here, we present tunable fibrous polyisocyanide (PIC) hydrogels functionalized with integrin-binding RGD peptides, cadherin-mimetic HAVDI peptides, or no ligands to direct mesenchymal, hybrid, or amoeboid-like migration of human adipose-derived stem cells without altering matrix mechanics. Using live-cell tracking, 3D displacement microscopy, matrix remodeling analysis, and YAP nuclear localization, we show that ligand identity governs adhesion organization, force transmission, and mechanotransduction. RGD-functionalized matrices promote {beta}1-integrin clustering, extensive matrix remodeling, strong YAP activation and upregulation of migration-related genes. In contrast, non-adhesive matrices limit adhesion formation, resulting in weak force transmission and amoeboid-like behavior. HAVDI-functionalized matrices induce cadherin clustering and heterogeneous cellular responses, indicating that a hybrid migration mode arises from adhesion organization rather than a distinct transcriptional program. Together, these findings demonstrate that ligand identity alone is sufficient to program migration mode in a force-responsive 3D matrix and provide a versatile platform to dissect cell-matrix interactions in complex environments. Statement of significanceO_LICell migration in tissues is highly adaptable, yet precise control of migration modes in defined 3D biomaterials remains challenging. C_LIO_LIWe introduce fibrous PIC hydrogels presenting RGD, HAVDI, or no adhesive ligands to bias human stem cells toward mesenchymal-like, hybrid, or amoeboid-like migration states. C_LIO_LIBy linking ligand identity to adhesion organization, matrix remodeling, YAP mechanotransduction, and gene expression, this work provides a minimal platform to dissect and engineer 3D cell-matrix interactions C_LI

Liquid crystalline mesophases exhibit structurally programmable internal architectures that enable co-loading of chemically orthogonal molecules within a single composite material. Realizing the potential of these materials for drug delivery requires a quantitative understanding of how tuning the composition affects internal mesophase architecture and consequently performance metrics such as payload release. Here, Flash NanoPrecipitation with hydrophobic ion pairing is used to prepare nanocarriers containing liquid crystalline mesophases co-encapsulating two compounds from widely different chemical classes: hydrophilic polymyxin B (logP -6) with one of four hydrophobic co-core materials (logP 7-11), achieving >75% encapsulation efficiency and up to 32% and 50% mass loadings for polymyxin and co-core. Synchrotron SAXS is used to quantify characteristic mesophase repeat spacing, which is found to be tunable as a function of composition. A strong correlation between d-spacing and polymyxin release rate is presented. Co-core chemistry and weight fraction jointly govern mesophase architecture, and repeat distance emerges as a structural metric linking these to the hydrophilic payload release kinetics. Mucus diffusivity and antibacterial efficacy are assessed as independent performance metrics, and results corroborate the release behavior. These findings establish a quantitative framework connecting material composition, mesophase architecture, and functional performance that can be applied toward rational co-formulation design. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/734853v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1d722eborg.highwire.dtl.DTLVardef@135adb7org.highwire.dtl.DTLVardef@11fe29eorg.highwire.dtl.DTLVardef@571165_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOToC Graphic TextC_FLOATNO Flash NanoPrecipitation yields liquid crystalline nanocarriers co-encapsulating with high efficiency payloads with widely distinct physicochemical properties. Synchrotron SAXS establishes characteristic repeat spacing as a quantitative structural metric directly governing hydrophilic release kinetics, providing a rational design framework linking mesophase architecture to functional performance across a range of payload structures. C_FIG

Amyloid fibrils are traditionally associated with protein misfolding disorders; however, increasing evidence indicates that they can also perform beneficial biological functions, including antimicrobial defense. Here, we investigated whether structurally distinct amyloid polymorphs of hen egg white lysozyme (HEWL) exhibit enhanced antibacterial activity compared with the native protein. HEWL was converted into two amyloid polymorphs, flexible fibrils (FFs) and rigid fibrils (RFs), and their antibacterial activities were evaluated against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacteria Escherichia coli (Top10) and Salmonella Typhimurium. Fibril formation was confirmed by circular dichroism (CD) spectroscopy, thioflavin T (ThT) fluorescence, and transmission electron microscopy (TEM), demonstrating morphologically distinct amyloid assemblies with different secondary-structure organizations. Fluorescence-based bacterial growth assays showed that native HEWL exhibited only moderate antibacterial activity, whereas both amyloid polymorphs produced potent, concentration-dependent bacterial growth inhibition. FFs and RFs consistently displayed greater antibacterial efficacy than native HEWL across all tested strains, with FFs exhibiting slightly stronger activity against S. Typhimurium. At concentrations of 600-800 M, FFs achieved >90% growth inhibition for all bacterial species examined. Cytotoxicity studies using SH-SY5Y human neuroblastoma cells demonstrated minimal toxicity for native HEWL, modest effects for FFs, and substantially greater toxicity for RFs, indicating that amyloid polymorphism influences both antimicrobial activity and mammalian cell compatibility. Collectively, these findings establish a direct relationship between amyloid structure, antibacterial efficacy, and cytotoxicity. The combination of potent antibacterial activity and relatively low cytotoxicity identifies FFs as a promising functional amyloid biomaterial for the development of next-generation antimicrobial materials.

Post-traumatic osteoarthritis (PTOA) is a progressive joint disease for which no disease-modifying osteoarthritis drugs (DMOADs) have been approved. Although injectable drug delivery systems can prolong therapeutic retention within the joint, it remains unclear whether local drug release kinetics influence disease-modifying efficacy. Here, we developed a modular platform of injectable supramolecular hydrogels using biocompatible, generally recognized as safe (GRAS) amphiphilic molecules and systematically engineered a range of degradation and drug release profiles. Using the cathepsin-K inhibitor L-006235 as a model DMOAD, we generated hydrogels with distinct release kinetics and evaluated their therapeutic performance in PTOA. Hydrogels exhibiting slower degradation and more sustained drug release like Sucrose Stearate (SS hydrogel) showed prolonged intra-articular retention and improved therapeutic outcomes. In a destabilization of the medial meniscus (DMM) mouse model, sustained-release formulations significantly reduced cartilage degeneration, preserved aggrecan expression, improved joint histopathology, and enabled effective monthly dosing. In contrast, formulations with faster degradation and release kinetics required more frequent administration to achieve comparable benefits. To our knowledge, this is the first study to establish local drug release kinetics as a critical determinant of disease-modifying efficacy in PTOA. This work provides one of the clearest demonstrations to date that engineering intra-articular release kinetics, rather than merely prolonging residence time, can improve disease-modifying outcomes. Our findings establish local release kinetics as a key design parameter for osteoarthritis therapeutics and highlight the potential of tunable supramolecular hydrogels for long-acting drug delivery.