Biomacromolecules

We probe the structural and rheological properties of ionically crosslinked alginate, a model biopolymer hydrogel, using microscopy, rheology and viscosity dependent molecular probes. This combination of techniques enables the quantification and correlation of microstructure, microviscosity, bulk viscoelasticity and yielding dynamics. By adjusting the stoichiometric ratio, R, between the alginate biopolymer and its cation crosslinks, we observe a transition from a homogeneous network-like structure to a coarse bundle-like structure at high (R>0.67) stoichiometric ratio. Intriguingly, these bundle-like structures have distinct and counter-intuitive rheological properties. Using molecular probes, we observe a continuous decrease in microviscosity that is correlated with a decrease in bulk elastic modulus and an increase in energy dissipation. This is accompanied by a transition in the hydrogel yielding under strain from a sharp, well-defined yield point to a continuous ductile-like yielding. We ascribe these surprising transitions to the looser intermolecular interaction between alginate biopolymers in the bundle-like state, as previously predicted by x-ray scattering experiments. These findings reveal new and counter-intuitive structure-property relations that demonstrate high crosslink concentration does not necessarily translate to optimal mechanical performance. SignificanceAlginate is a polysaccharide biopolymer naturally found in brown seaweed cell walls. Extracted alginate forms ionically crosslinked hydrogels that are strong, flexible and increasingly valuable in the biomedical, packaging and food industries. A large part of the utility of these hydrogels stems from the ease with which their mechanics can be tuned through adjusting the stoichiometry between alginate and its ionic crosslinks. However, little is known about how the material properties of alginate hydrogels, in particular their rheology and dynamics, are affected by microscale structural transitions at high stoichiometric ratios. Here, we use a multi-modal approach to describe and correlate hydrogel material properties and to demonstrate that increased polymer crosslinking can, counterintuitively, sometimes weaken hydrogel performance.

Hydrogels are widely used in sensing, delivery, and tissue engineering because their transport properties can be tuned through material design. However, while hydrogel permeability is often characterized using small molecules, many practical applications depend on the uptake and retention of much larger species, including protein conjugates and nanoparticles. Here, we systematically investigate how polyethylene glycol (PEG)-acrylate hydrogel microparticle formulation influences accumulation of signal-generating probes spanning a broad size range. We fabricated particles across a 36-condition design space varying nominal PEG-acrylate molecular weight, polymer weight percent, and UV crosslinking dose, and related formulation-dependent probe accumulation to particle swelling behavior. Increasing nominal PEG-acrylate molecular weight and decreasing polymer weight percent produced more highly swollen particles and strongly enhanced accumulation of fluorescent streptavidin conjugates, with the largest effects observed for bulky labels such as allophycocyanin and phycoerythrin. Gold nanoparticle accumulation was even more formulation-restricted, with detectable colorimetric signal observed primarily in the most permissive formulations. These findings establish design rules linking PEG hydrogel formulation to size-dependent accumulation and show that formulations suitable for small probes may be inadequate for larger reporters. More broadly, this framework may inform the design of hydrogels for particle-based assays as well as other applications where transport of macromolecules or nanoscale materials is important.

The recent development of Volumetric Imaging via Photochemical Sectioning (VIPS) has enabled nanoscale imaging of whole-mount tissue samples of virtually any size by embedding intact tissue in a photocleavable, superabsorbent hydrogel. However, the efficacy of sample embedding, imaging, and photochemical sectioning is fundamentally governed by the mechanical stiffness, structural stability, and photodegradation kinetics of the photocleavable hydrogel (PC-gel) polymer network. To elucidate the effect of the photosensitive crosslinker design on these critical properties, we synthesized a set of photocleavable crosslinkers (PCs) with varying polyethylene glycol (PEG) backbone lengths and prepared the corresponding PC-gels under a fixed monomer formulation and polymerization condition. We quantified and compared the viscoelastic properties of the formed PC-gels at their swollen states, and found that the crosslinker length markedly reshaped the PC-gel mechanics. In addition, we evaluated the light-triggered degradation of the PC-gels using both wide-field and spatially-controlled illumination. We found that PC-1000, PC-1500, and PC-2000 gels remained comparably photodegradable, all enabling on-demand, spatially confined decrosslinking under such illuminations. These results provide practical guidelines for modulating the crosslinker architecture of PC-gel polymer networks to achieve optimal physicochemical properties for whole-mount tissue imaging using VIPS.

Proteases are an important class of proteolytic enzymes having great importance in both basic science and industrial applications. While cells tightly regulate the spatio-temporal activity of different proteases for cellular homeostasis, mis-regulation often leads to adverse effects. In this context, the protease activity of papain and its activation by L-cysteine is poorly understood in the literature. Herein, we discover that the protease activity of papain can be effectively regulated via a spontaneous liquid-liquid phase separation (LLPS) pathway. We show that papain undergoes biomolecular condensation via spontaneous LLPS under macromolecular crowding through the involvement of intermolecular hydrophobic interactions. Secondary structure analyses revealed a compact conformation of phase-separated papain with increased -helix content. Although native free papain is found to be active towards synthetic and protein substrates, the proteolytic digestion produces heterogeneous peptide aggregates. In contrast, we found that papain droplets remain dormant toward protein digestion due to the disulfide linkage of the active cysteine residue (Cys-25) in its compact conformational state. More importantly, we show that the protease activity of phase-separated papain can be reactivated in the presence of L-cysteine to produce uniform soluble peptide fragments. Our findings indicate that although disulfide linkages are not necessary for the phase separation of papain, upon phase separation, intermolecular interactions between phase-separated papain result in the formation of disulfide linkages involving active Cys-25 residues. The present discovery has tremendous technological importance to boost the efficacy of meat tenderization in the food industry.

The High Mobility Group Box 1 (HMGB1) protein performs multiple essential functions in the body, ranging from DNA regulation to the activation and mediation of immune responses. However, HMGB1 has been also implicated in several pathological conditions, such as rheumatoid arthritis, sepsis, autoimmune diseases, tumors, and Alzheimer's disease. As a result, HMGB1 is of increasing interest as a therapeutic target. Binding to heparin has been reported to inhibit HMGB1's pathological activity during sepsis in clinical settings. In this work, we compare the interactions of HMGB1 with heparin and its' synthetic analog linear polyglycerol sulfate (lPGS) from the viewpoint of stability and changes to association behavior. This analysis focuses on thermal stability, secondary-structure changes, and particle-size evolution using nano-differential scanning fluorimetry (nanoDSF), circular dichroism spectroscopy (CD), and dynamic light scattering (DLS).

Amyloid fibrils are highly ordered protein aggregates characterized by a conserved cross-{beta}-sheet architecture despite originating from structurally diverse precursor proteins. Growing evidence suggests that interactions between different amyloidogenic proteins can modulate aggregation pathways through heterologous cross-seeding; however, the influence of seed polymorphism on the structure and biological properties of cross-seeded fibrils remains poorly understood. Here, we investigated the cross-seeding of native human insulin by two structurally distinct polymorphs of hen egg-white lysozyme (HEWL): flexible fibrils (FFs) and rigid fibrils (RFs). Native insulin remained stable under physiological conditions and underwent spontaneous fibrillation only under acidic conditions. In contrast, both HEWL polymorphs efficiently induced insulin aggregation at physiological pH, bypassing the nucleation barrier. Thioflavin T fluorescence, circular dichroism spectroscopy, and transmission electron microscopy revealed that lysozyme FFs templated the formation of insulin flexible fibrils (IFFs), whereas lysozyme RFs produced insulin rigid fibrils (IRFs), demonstrating that the structural characteristics of the parental HEWL polymorphs were propagated during heterologous cross-seeding. The toxicity of the resulting insulin fibrils was evaluated in SH-SY5Y neuronal cells and CCF-STTG1 astrocytes. IFFs exhibited minimal cytotoxicity and only subtle morphological alterations, whereas IRFs caused modest reductions in cell viability accompanied by more pronounced cellular damage. These findings demonstrate that the structural polymorphism of HEWL fibrils governs both the architecture and biological activity of cross-seeded insulin fibrils, highlighting amyloid polymorphism as an important determinant of heterologous amyloid propagation and a potential design principle for engineering functional amyloid-based biomaterials and protein delivery platforms.

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.

Tryptophan is a biologically important redox-active amino acid whose functions in proteins, including long-range electron transfer, protection against oxidative damage, and environmental sensing, are governed not only by its chemical identity but also by its precise structural organization. Inspired by this biological principle, we investigated whether controlling the organization of tryptophan within crystalline materials could provide a strategy for modulating its physicochemical properties and molecular accessibility. Using identical molecular components but distinct assembly pathways, tryptophan was organized either as a confined guest within a preformed Zn-imidazolate framework, yielding a star-shaped crystalline architecture, or as an integral coordination component during framework growth, producing a distinct layered Zn- tryptophan crystalline framework. Although assembled from the same building blocks, these two organization modes generated fundamentally different crystal structures, morphologies, and mechanisms of biomolecule incorporation. In both architectures, incorporation of tryptophan into the crystalline environment preserved its intrinsic fluorescence while producing robust fluorescence under multiple excitation wavelengths, highlighting the strong influence of molecular organization on its optical response. The structural modes also exhibited distinct encapsulation efficiencies and pH-dependent molecular accessibility, while secondary calcium-alginate fixation provided an additional level of control over guest retention without disrupting the underlying crystalline architecture. These results demonstrate that engineering the structural organization of tryptophan provides a versatile strategy for tuning the optical behavior, molecular accessibility, and functional integration of a biologically important redox-active amino acid in crystalline materials, establishing a foundation for future biomimetic redox architectures, responsive sensing platforms, and controlled molecular delivery.

1.Surface functionality and pH play a decisive role in governing the structural dynamics, hydration, and drug-binding behaviour of dendrimers. Here, all-atom molecular dynamics (MD) simulations were performed on five generations of PAMAM (G1-G5) and PETIM (G2-G6) dendrimers with O-core and N-core architectures, functionalized with amine, carboxylic acid, or sugar terminal groups under different protonation states. Protonation of the tertiary branch-point amines expands the dendrimer structure, increases internal porosity and hydration, and enhances structural fluctuations across both families. In contrast, non-protonated amine -NH2 (NP) and carboxylic acid -COOH (NP) terminated dendrimers, together with deprotonated carboxylate-COO- (DeP) systems, retain comparatively compact conformations. Sugar-functionalized dendrimers ({beta}-galactose-terminated PETIM and D-glucose-terminated PAMAM) are most hydrated and structurally rigid, whereas amine-terminated dendrimers exhibit the greatest conformational dynamics. PAMAM dendrimers with -NH2, -NH3+, and -COO- terminal groups are generally more hydrated than their PETIM counterparts. However, {beta}-galactose-terminated PETIM dendrimers are more hydrophilic than D-glucose-terminated PAMAM dendrimers. N-core PETIM dendrimers also adopt more compact and spherical conformations than equivalent O-core PETIM dendrimers. Drug-binding MD simulations show that curcumin binding is dominated by van der Waals (vdW) interactions, whereas doxorubicin complexation is primarily driven by electrostatic interactions. Among the investigated surface functionalities, -NH2 (NP), -NH3+ (P), -COOH (NP), and -COO- (DeP) terminations exhibit the most favourable drug-binding characteristics. Except for deprotonated carboxylate systems, curcumin binds more strongly than doxorubicin. Overall, these findings establish molecular-level relationships between surface functionality, protonation state, dendrimer architecture, and drug-binding behaviour, providing design principles for pH-responsive dendrimer nanocarriers with enhanced drug-loading and controlled-release performance. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/742721v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@119bf29org.highwire.dtl.DTLVardef@1554d86org.highwire.dtl.DTLVardef@154a254org.highwire.dtl.DTLVardef@16d5c5b_HPS_FORMAT_FIGEXP M_FIG C_FIG

The thermal stability of collagen triple helices is strongly influenced by the amino acid sequence of the repeating Gly-X-Y tripeptides, yet how these residue-specific interactions are integrated within an extended triple helix to determine thermal behavior remains poorly understood. Here, we addressed this question using recombinant collagen mimetic peptides (rCMPs) containing extended native sequences from the 1(I) and 2(I) chains of human type I collagen. Triple-helix formation was nucleated by a C-terminal foldon domain and further stabilized by interchain disulfide crosslinking, allowing the apparent melting temperature (T) to reflect interactions within the triple-helical domain independent of nucleation. The stabilizing effects of Pro and Y-position Arg identified in host-guest peptides were largely preserved in extended triple helices, whereas the proposed Lys-Gly-Glu (KGE) interchain salt bridge produced little measurable stabilization, demonstrating the influence of sequence context. Remarkably, identical triple-helical sequences exhibited markedly different thermal behavior when unfolding was initiated under different conditions. Nevertheless, extended triple helices differing substantially in sequence and length retained an apparently two-state thermal transition. These findings support a mechanism in which unfolding is preferentially initiated within regions of lower intrinsic stability, while the continuity of the triple helix couples neighboring regions into a cooperative unfolding process throughout the helix. This mechanism provides a plausible explanation for the longstanding paradox that extended collagen triple helices exhibit persistent sequence-dependent thermodynamic heterogeneity despite a two-state thermal transition, and a framework for investigating how sequence-dependent stability contributes to the structure and function of collagen molecules. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/741348v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@f311aorg.highwire.dtl.DTLVardef@160dc76org.highwire.dtl.DTLVardef@29e989org.highwire.dtl.DTLVardef@1a3279c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Articular cartilage damage often progresses to osteoarthritis (OA), a degenerative joint disease characterized by chronic pain, limited mobility, and reduced quality of life. Tissue engineering approaches using poly (ethylene glycol) (PEG)-based hydrogels offer tunable mechanical properties and bioactive functionalization, yet the influence of surface charge on cartilage regeneration remains underexplored. Moreover, recombinant fibroblast growth factor 18 (FGF18) has successfully improved cartilage tissue thickness in clinical trials, but required high dosages and recurring injections may limit compliance. Here, we developed a granular microgel-based platform with bioorthogonally tethered FGF18 to evaluate the interplay of microgel surface charge and growth factor presentation on chondrogenesis. Azide groups were incorporated onto the microgel surfaces to enable site specific FGF18 conjugation across microgel scaffolds with distinct surface charges. When seeded with mesenchymal stromal cells, microgel scaffolds functionalized with FGF18 outperformed their unmodified counterparts, evidenced by higher GAG content, collagen content, and compressive modulus. In a murine microfracture model, anionic and zwitterionic microgel scaffolds tethered with FGF18 increased cartilage regeneration compared to nonionic microgels. We detected increased collagen II content within defects treated with tethered FGF18 microgel scaffolds. This work demonstrates the role of surface charge and growth factor presentation in directing cell behavior and tissue repair, advancing the design of biomaterials for cartilage regeneration. HIGHLIGHTSO_LICovalent tethering of FGF18 to microgel surface enables localized bioactivity C_LIO_LIFGF18 tethered microgel scaffolds enhance extracellular matrix deposition and chondrogenic differentiation of murine mesenchymal stromal cells in vitro C_LIO_LILocalized FGF18 presentation improves cartilage tissue formation and mechanical properties in vivo, with anionic and zwitterionic microgel scaffolds outperforming nonionic scaffolds C_LIO_LIFGF18 presentation is a more potent stimulus than microgel surface charge for cartilage regeneration C_LI

The environmental impact of synthetic textiles has prompted the search for sustainable and biodegradable alternatives. This study correlates the growth conditions used to produce kombucha-derived cellulose non-woven mats with their mechanical performance as a function of post-processing. Systematically, the fermentation and growth parameters of the non-wovens, including inoculum density, carbon-source loading, temperature, and pH value were investigated. Thick, uniform non-wovens were obtained using mildly acidic conditions that balanced nutrient availability and growth rate, moderate inoculum and carbon loading at 30 {degrees}C. Next, we used uniaxial tensile testing and rheology to thoroughly compare the mechanical properties of two post-processing routes, lyophilization and oven-drying against the as-produced wet non-wovens. Overall, the lyophilized non-wovens displayed the highest ultimate tensile strength (14.36 {+/-}0.9 MPa) and elongation at break (24.54 {+/-}1.9%), which were statistically greater than the oven-dried (2.54 {+/-}0.3 MPa, 6.03 {+/-}0.8%) and the wet non-wovens (1.66 {+/-}0.3 MPa, 9.35 {+/-}2.8%). We conclude by performing a proof-of-concept recyclability experiment: we showed that kombucha-derived clothing could be enzymatically degraded and then re-manufactured into new nanofibers by electrospinning. Together, these results demonstrate a circular pathway encompassing the growth and processing of mechanically robust kombucha-derived cellulose non-wovens, as well as their biodegradation and re-manufacturing.

The chemical synthesis of well-defined, self-assembling semi-synthetic proteins (SSPs) has attracted growing interest in recent years. Approaches such as micelle-assisted protein-labeling technology (MAPLabTech) and supramolecule-assisted protein-labeling technology (SAPLabTech) have been used to generate a wide range of SSPs. The central challenge in synthesizing SSPs is solubilizing a hydrophobic chemical probe in aqueous medium prior to bioconjugation. Both MAPLabTech and SAPLabTech rely on non-covalent interactions to solubilize hydrophobic probes and present certain limitations. The present study introduces a complementary chemical strategy in which a hydrophobic chemical probe is covalently tagged with a cleavable, water-soluble dendritic domain. This covalent tagging renders the probe fully water-soluble, enabling quantitative bioconjugation to yield monomeric semi-synthetic proteins. Subsequent, selective removal of the solubility tag converts the hydrophilic semi-synthetic proteins into facially amphiphilic, semi-synthetic proteins.

Water-soluble polymers commonly interact with cell membranes, but their interactions are poorly understood. Here, we investigate polyethylene glycol (PEG) and dextran (DEX) interactions with different model lipid membranes. Using total internal reflection fluorescence microscopy, we observe that PEG and DEX trigger strikingly different membrane responses - DEX induces extensive membrane remodeling, including localized multilamellar domain formation, while PEG does not. Combining fluorescence spectroscopy, fluorescence anisotropy, and vibrational sum frequency spectroscopy, we show that DEX perturbs lipid headgroup hydration by displacing interfacial water with minimal effects on lipid packing, while PEG largely preserves this hydration layer. We find that membrane binding affinity alone does not determine the extent to which hydrophilic polymers perturb membrane structure and interfacial properties; and that lipid headgroup hydration, rather than lipid charge, is a general regulator of hydrophilic polymer-membrane interactions. This work gives mechanistic insights into how neutral polymers interact with cells and vesicles, with relevance to cell biology and drug delivery. O_FIG O_LINKSMALLFIG WIDTH=169 HEIGHT=200 SRC="FIGDIR/small/739366v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1d1c6corg.highwire.dtl.DTLVardef@135361corg.highwire.dtl.DTLVardef@73970corg.highwire.dtl.DTLVardef@c19615_HPS_FORMAT_FIGEXP M_FIG C_FIG

Antibody drug conjugates (ADCs) are a burgeoning class of targeted therapies. However, limitations in their synthesis and efficiency of payload delivery restrict their clinical utility. Here we report a supramolecular assembly (SMA) ADC conjugation method, which allows site-specific, uniform drug loading, resulting in an enhanced pharmacokinetic profile and in vivo efficacy. This peptide conjugation strategy relies on spontaneous heterotetrameric coiled-coil formation between a pair of peptides appended on the C-terminus and a drug-loaded complementary pair in aqueous solution. Pairing this SMA conjugation with an antibody that targets the dual-endothlin-1/VEGF signal peptide receptor (DEspR), a pancreatic ductal adenocarcinoma (PDAC) specific receptor, retains antibody binding and plasma stability. When the anti-DEspR monoclonal antibody is conjugated with monomethyl auristatin E (MMAE), the ensuing ADC internalizes following cell surface binding and induces selected cell death in multiple DEspR positive PDAC cell lines. In vivo, the ADC exhibits favorable pharmacokinetics, high tumor specificity, and improves overall survival in a rat orthotopic model of pancreatic peritoneal carcinomatosis, compared to conventional ADC conjugation. A heterotetrameric coiled-coil structure enables the efficient synthesis of a potent ADC, further documenting the versatility of supramolecular scaffolds as key orthogonal building block for site-specific conjugation in biopharmaceutical and biomaterial drug delivery systems. One Sentence SummaryCombining site-specific conjugation of monomethyl auristatin E, via the use of biologically inspired heterotetrameric coiled-coils, with a tumor-selective antibody targeting the dual-endothlin-1/VEGF signal peptide receptor affords a highly effective, ADC, which improves survival in a rat orthotopic model of pancreatic peritoneal carcinomatosis compared to standard cysteine-conjugated analogues with higher drug loading.

Thioamides are natural post-translational modifications of the peptide backbone and can be introduced synthetically to probe protein folding or functionalize peptides for translational applications. In this work, we demonstrate that thioamide-containing peptides with C-terminal thioesters can be efficiently generated using Knorr pyrazole activation and used in subsequent native chemical ligation reactions to generate thioamide containing proteins. We compare this method to acyl azide activation and find that both routes provide similar yields. We also investigate ultrasound-mediated desulfurization of the ligation site cysteine for potential advantages over chemical radical initiators. Scaling up our syntheses allows us to study thioamide perturbations to the {beta}-sheet region of the B1 domain of protein G (GB1) as well as {beta}-strand interactions in amyloid fibrils of the Parkinsons disease protein -synuclein. In both contexts, we observe dramatic destabilization of the {beta}-sheet networks, manifested in decreased GB1 thermal stability and altered folding and slowed aggregation of -synuclein. These findings illustrate the impact that a single atom substitution can have on cooperative hydrogen bonding networks and prompt future study of both systems.

DNA origami nanoparticles (DONs) are attractive nanocarriers of controllable size, shape and addressability that have potential for treating eye diseases by overcoming ocular barriers. However, suboptimal physiological stability and poor cell uptake due to the negative charge may limit their use. Previous reports show that electrostatic complexation of DONs with cationic PEG-oligolysine block-copolymers like PEG5K-K10 can improve structural integrity and promote cell internalization. Here, we investigated a dual approach of PEG5K-K10 coatings and PL3 targeting peptides to improve uptake of 24-helix bundle (24HB) DONs into Y-79 retinoblastoma cells. Uptake studies revealed that PEG5K-K10 was essential for DON uptake in Y-79 cells, as uptake only occurred upon exceeding a distinct PEG5K-K10 amount. Longer exposure times or increased polymer amounts improved cell association. However, no beneficial effect of PL3 was observed. While free PEG5K-K10 reduced cell viability at higher concentrations (IC50 36.8 {micro}M), coated DONs were well-tolerated. Furthermore, single particle tracking in ex vivo porcine eyes revealed comparable vitreal mobility for uncoated and coated 24HB, with a slight decrease at higher coating amounts. Our findings highlight that PEG5K-K10 can enhance ocular cell uptake without limiting nanoparticle diffusivity in the vitreous, and support further optimization of DONs for ocular drug delivery.

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.

Bioxolography enables high-resolution fabrication of geometrically complex, cell-laden constructs for tissue engineering. However, tissue-relevant cell densities conflict with the optical transparency required for efficient dual-color volumetric printing. In this work, we extend the Bioxolography toolbox to include refractive index (RI) matching for cell-laden bioresins using iodixanol (IDX). Remarkably, IDX enhances optical transparency and boosts reactivity -- a phenomenon unique to Xolography. Yet, excessive IDX compromises dual-color efficiency through increased absorption and undesired UV-only curing, underscoring a central trade-off between optical clarity and photochemical performance. Systematic tuning of resin compositions along an iso-refractive index line demonstrated the versatility of Bioxolography, with IDX enhancing polymerization and 4-Hydroxy-TEMPO providing biocompatible inhibition. Optimizing composition and printing parameters yielded GelMA hydrogels with cell densities up to 5{middle dot}106 cells{middle dot}mL-1. Cell-laden prints achieved sub-100 {micro}m resolution and complex geometries such as channels and gyroids. Using skeletal muscle tissue as a model, we validated RI matched Bioxolography as a promising strategy for tissue engineering by demonstrating cell alignment along printed grooves and formation of mature muscle fibers characterized by MyoHC+ staining and fusion index. By integrating physical, chemical, and biological perspectives, this work advances Xolography toward biomaterials development and reinforces its position as an emerging volumetric (bio)printing technology. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/729865v1_ufig1.gif" ALT="Figure 1"> View larger version (86K): org.highwire.dtl.DTLVardef@407d86org.highwire.dtl.DTLVardef@1f116b4org.highwire.dtl.DTLVardef@109a437org.highwire.dtl.DTLVardef@1fc4ece_HPS_FORMAT_FIGEXP M_FIG C_FIG For printing higher cell density bioresins with Xolography, iodixanol (IDX) is added for refractive index-matching. The addition leads to an unexpected additional effect with increased reactivity in the dual-color photopolymerization. With careful adjustment of the resin composition and the printing parameters, Bioxolography is proven as a viable tool for tissue engineering.

One crucial step during early osteogenesis is the embedding of osteoblasts within a collagen-rich extracellular matrix (osteoid), where they subsequently differentiate into a functional network of osteocytes. However, reconstructing 3D osteocyte networks in vitro remains a major challenge. We recently developed a synthetic microporous hydrogel to support the in vitro culture of 3D bone cell networks. Although matrix biodegradability facilitates cell-material interactions, the influence of micropores on bone tissue morphogenesis and differentiation remains poorly understood. Here, we investigate the effect of cell-scale micropores on bone cell morphogenesis and osteogenic differentiation in vitro. By exploiting polymerization-induced phase separation (PIPS) between 4-arm polyethylene glycol vinyl sulfones and dextran in the presence of hyaluronan, we generated matrix metalloproteinase-sensitive hydrogels with cell-scale micropores. Increasing the dextran concentration enlarged the average pore size from 4 m to 8 m, accompanied by a slight decrease in mechanical stiffness. Following encapsulation within these hydrogels, primary human osteoblasts remained highly viable. Hydrogels with larger pores supported extensive 3D cell network formation, whereas hydrogels with smaller pores exhibited enhanced osteogenic differentiation following 21 days of osteogenic culture. Together, these findings highlight that bone cells are sensitive to microporous physical cues and even minor changes over pore sizes can make an impact on osteocyte-like morphogenesis and differentiation in vitro.