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We report a new strategy that allows reversible tuning of the stiffness and stress-relaxation of viscoelastic hydrogels cross-linked via hydrazone bonds by incorporating a small-molecule competitor. The competitor molecule competes for the formation of reversible hydrazone bonds and temporarily reduces the cross-linking density in the hydrogel, thus softening the hydrogel and accelerating its stress-relaxation. By rapidly diffusing the competitor in and out of the hydrogel, the mechanical properties of hydrogels can be reversibly altered over many cycles. We further examined the biocompatibility of the competitor and explored its application in cell delivery via injection by temporarily adjusting the hydrogel mechanical properties to improve cell viability during the injection.

Three-dimensional bioprinting has emerged as a promising tool for spatially patterning cells to fabricate models of human tissue. Here, we present an engineered bioink material designed to have viscoelastic mechanical behavior, similar to that of living tissue. This viscoelastic bioink is cross-linked through dynamic covalent bonds, a reversible bond type that allows for cellular remodeling over time. Viscoelastic materials are challenging to use as inks, as one must tune the kinetics of the dynamic cross-links to allow for both extrudability and long-term stability. We overcome this challenge through the use of small molecule catalysts and competitors that temporarily modulate the cross-linking kinetics and degree of network formation. These inks were then used to print a model of breast cancer cell invasion, where the inclusion of dynamic cross-links was found to be required for the formation of invasive protrusions. Together, we demonstrate the power of engineered, dynamic bioinks to recapitulate the native cellular microenvironment for disease modeling.

Disruption of the tumor extracellular matrix (ECM) may alter immune cell infiltration into the tumor and antitumor T cell priming in the tumor-draining lymph nodes (tdLNs). Here, we explore how intratumoral enzyme treatment (ET) of B16 melanoma tumors with ECM-depleting enzyme hyaluronidase alters adaptive and innate immune populations, including T cells, DCs, and macrophages, in the tumors and tdLNs. ET increased CD103

We systematically investigate model synthetic and natural bottlebrush polyelectrolyte solutions through an array of experimental techniques (osmometry and neutron and dynamic light scattering) along with molecular dynamics simulations to characterize and contrast their structures over a wide range of spatial and time scales. In particular, we perform measurements on solutions of aggrecan and the synthetic bottlebrush polymer, poly(sodium acrylate), and simulations of solutions of highly coarse-grained charged bottlebrush molecules having different degrees of side-branch density and inclusion of an explicit solvent and ion hydration effects. While both systems exhibit a general tendency toward supramolecular organization in solution, bottlebrush poly(sodium acrylate) solutions exhibit a distinctive "polyelectrolyte peak" in their structure factor, but no such peak is observed in aggrecan solutions. This qualitative difference in scattering properties, and thus polyelectrolyte solution organization, is attributed to a concerted effect of the bottlebrush polymer topology and the solvation of the polymer backbone and counterions. The coupling of the polyelectrolyte topological structure with the counterion distribution about the charged polymer molecules along with direct polymer segmental hydration makes their solution organization and properties "tunable," a phenomenon that has significant ramifications for biological function and disease as well as for numerous materials applications.

The ionic complexation of polyelectrolytes is an important mechanism underlying many important biological processes and technical applications. The main driving force for complexation is electrostatic, which is known to be affected by the local polarity near charge centers, but the impact of which on the complexation of polyelectrolytes remains poorly explored. We developed a homologous series of well-defined polyelectrolytes with identical backbone structures, controlled molecular weights, and tunable local polarity to modulate the solvation environment near charged groups. A multitude of systematic, accurate phase diagrams were obtained by spectroscopic measurements of polymer concentrations via fluorescent labeling of polycations. These phase diagrams unambiguously revealed that the liquidlike coacervation is more stable against salt addition at reduced local polarity over a wide range of molecular weights. These trends were quantitatively captured by a theory of complexation that incorporates the effects of dispersion interactions, charge connectivity, and reversible ion-binding, providing the microscopic design rules for tuning molecular parameters and local polarity., Coacervation diagrams from well-defined homologous polyelectrolyte series revealed impacts of local polarity near charge groups and were quantitatively captured by a minimal theoretical model.

Human intestinal organoids from primary human tissues have the potential to revolutionize personalized medicine and preclinical gastrointestinal disease models. A tunable, fully defined, designer matrix, termed hyaluronan elastin‐like protein (HELP) is reported, which enables the formation, differentiation, and passaging of adult primary tissue‐derived, epithelial‐only intestinal organoids. HELP enables the encapsulation of dissociated patient‐derived cells, which then undergo proliferation and formation of enteroids, spherical structures with polarized internal lumens. After 12 rounds of passaging, enteroid growth in HELP materials is found to be statistically similar to that in animal‐derived matrices. HELP materials also support the differentiation of human enteroids into mature intestinal cell subtypes. HELP matrices allow stiffness, stress relaxation rate, and integrin‐ligand concentration to be independently and quantitatively specified, enabling fundamental studies of organoid–matrix interactions and potential patient‐specific optimization. Organoid formation in HELP materials is most robust in gels with stiffer moduli (G’ ≈ 1 kPa), slower stress relaxation rate (t 1/2 ≈ 18 h), and higher integrin ligand concentration (0.5 × 10−3–1 × 10−3 m RGD peptide). This material provides a promising in vitro model for further understanding intestinal development and disease in humans and a reproducible, biodegradable, minimal matrix with no animal‐derived products or synthetic polyethylene glycol for potential clinical translation., A tunable, designer matrix, termed hyaluronan elastin‐like protein (HELP) that enables the formation, differentiation, and passaging of adult primary tissue‐derived organoids is reported. HELP matrices allow stiffness, stress relaxation rate, and integrin‐ligand concentration to be independently and quantitatively specified, enabling fundamental studies of organoid–matrix interactions and potential patient‐specific optimization.

Correction for ‘Actuated 3D microgels for single cell mechanobiology’ by Berna Özkale et al., Lab Chip, 2022, 22, 1962–1970, https://doi.org/10.1039/D2LC00203E.

There is a great interest in the synthesis and characterization of polyelectrolytes that mimic naturally occurring bottlebrush polyelectrolytes to capitalize on the unique properties of this class of macromolecules. Charged bottlebrush polymers form the protective mucus layer in the lungs, stomach, and orifices of animals and provide osmotic stabilization and lubrication to joints. In the present work, we systematically investigate bottlebrush poly(sodium acrylates) through a combination of measurements of solution properties (osmometry, small-angle neutron scattering, and dynamic light scattering) and molecular dynamics simulations, where the bottlebrush properties are compared in each case to their linear polymer counterparts. These complementary experimental and computational methods probe vastly different length- and timescales, allowing for a comprehensive characterization of the supermolecular structure and dynamics of synthetic polyelectrolyte bottlebrush molecules in solution.

The principles governing ion and macroion partitioning in nonstoichiometric coacervates are elucidated., A wide variety of intracellular membraneless compartments are formed via liquid-liquid phase separation of charged proteins and nucleic acids. Understanding the stability of these compartments, while accounting for the compositional heterogeneity intrinsic to cellular environments, poses a daunting challenge. We combined experimental and theoretical efforts to study the effects of nonstoichiometric mixing on coacervation behavior and accurately measured the concentrations of polyelectrolytes and small ions in the coacervate and supernatant phases. For synthetic polyacrylamides and polypeptides/DNA, with unequal mixing stoichiometry, we report a general “looping-in” phenomenon found around physiological salt concentrations, where the polymer concentrations in the coacervate initially increase with salt addition before subsequently decreasing. This looping-in behavior is captured by a molecular model that considers reversible ion binding and electrostatic interactions. Further analysis in the low-salt regime shows that the looping-in phenomenon originates from the translational entropy of counterions that are needed to neutralize nonstoichiometric coacervates.

The physical and architectural cues of the extracellular matrix (ECM) play a critical role in regulating important cellular functions such as spreading, migration, proliferation, and differentiation. Natural ECM is a complex viscoelastic scaffold composed of various distinct components that are often organized into a fibrillar microstructure. Hydrogels are frequently used as synthetic ECMs for 3D cell culture, but are typically elastic, due to covalent crosslinking, and non-fibrillar. Recent work has revealed the importance of stress relaxation in viscoelastic hydrogels in regulating biological processes such as spreading and differentiation, but these studies all utilize synthetic ECM hydrogels that are non-fibrillar. Key mechanotransduction events, such as focal adhesion formation, have only been observed in fibrillar networks in 3D culture to date. Here we present an interpenetrating network (IPN) hydrogel system based on HA crosslinked with dynamic covalent bonds and collagen I that captures the viscoelasticity and fibrillarity of ECM in tissues. The IPN hydrogels exhibit two distinct processes in stress relaxation, one from collagen and the other from HA crosslinking dynamics. Stress relaxation in the IPN hydrogels can be tuned by modulating HA crosslinker affinity, molecular weight of the HA, or HA concentration. Faster relaxation in the IPN hydrogels promotes cell spreading, fiber remodeling, and focal adhesion (FA) formation - behaviors often inhibited in other hydrogel-based materials in 3D culture. This study presents a new, broadly adaptable materials platform for mimicking key ECM features of viscoelasticity and fibrillarity in hydrogels for 3D cell culture and sheds light on how these mechanical and structural cues regulate cell behavior.

Rational design of dynamic hydrogels with desirable viscoelastic behaviors relies on an in-depth understanding of the principles correlating molecular parameters and macroscopic properties. To quantitatively elucidate such principles, a series of dynamic covalent hydrogels crosslinked via hydrazone bonds is designed. The exchange rate of the hydrazone bond is tuned by varying the concentration of an organic catalyst, while maintaining the crosslinking density unchanged. This strategy of independently tuning exchange dynamics of crosslinks and crosslinking density allows unambiguous analysis of the viscoelastic response of the dynamic hydrogels as a function of their network parameters. It is found that the terminal relaxation time of the dynamic hydrogels is primarily determined by two factors: the exchange rate of crosslinks and the number of effective crosslinks per polymer chain, and is independent of the network architecture. Furthermore, a universal correlation is identified between the terminal relaxation time determined from stress relaxation and the exchange rate determined via reaction kinetics, which can be generalized to any viscoelastic hydrogel network, in principle. This quantitative correlation facilitates the development of dynamic hydrogels with a variable desired viscoelastic response based on molecular design.

Hydrogels are commonly used as artificial extracellular matrices for 3D cell culture and for tissue engineering. Viscoelastic hydrogels with tunable stress relaxation have recently been developed, and stress relaxation in the hydrogels has been found to play a key role in regulating cell behaviors such as differentiation, spreading, and proliferation. Here we report a simple but precise materials approach to tuning stress relaxation of alginate hydrogels with polyethylene glycol (PEG) covalently grafted onto the alginate. Hydrogel relaxation was modulated independent of the initial elastic modulus by varying molecular weight and concentration of PEG along with calcium crosslinking of the alginate. Increased concentration and molecular weight of the PEG resulted in faster stress relaxation, a higher loss modulus, and increased creep. Interestingly, we found that stress relaxation of the hydrogels is determined by the total mass amount of PEG in the hydrogel, and not the molecular weight or concentration of PEG chains alone. We then evaluated the utility of these hydrogels for 3D cell culture. Faster relaxation in RGD-coupled alginate-PEG hydrogels led to increased spreading and proliferation of fibroblasts, and enhanced osteogenic differentiation of mesenchymal stem cells (MSCs). Thus, this work establishes a new materials approach to tuning stress relaxation in alginate hydrogels for 3D cell culture.

Remotely and locally triggered release of therapeutic species by X-ray irradiation is highly desired to enhance the efficacy of radiotherapy. However, the development of such X-ray responsive nanosystems remains a challenge, especially in response to high energy clinically relevant X-ray radiation. Herein, we report novel nitroimidazole ligated gold nanoparticles (AuNPs) that synergistically function to release nitrite, an important precursor for nitric oxide and reactive nitrogen species that sensitize cancer cells, upon radiation with clinically used 6 MeV X-rays, while no release was detected without radiation. These functional AuNPs were prepared with surface-grafted nitroimidazole as the nitrite-releasing agent, cell-penetrating peptide (CPP) to induce nucleus localization, and poly(ethylene glycol) for water solubility. In vitro radiotherapy using such nanoparticles showed enhanced sensitivity of hypoxic cancer cells to X-ray radiation, presumably due to the generation of both reactive oxygen and nitrogen species. The dose modifying factor (DMF) was found to be 0.71 for the dual-functionalized nanoparticle, which indicates that significant lower X-ray doses are required to achieve the same therapeutic effects. Thus, X-ray triggered nitrite release from gold-nitroimidazole nanosystems offers a novel strategy to sensitize cancer cells for improved radiotherapy.

Injectable and biocompatible hydrogels have become increasingly important for cell transplantation to provide mechanical protection of cells during injection and a stable scaffold for cell adhesion post-injection. Injectable hydrogels need to be easily pushed through a syringe needle and quickly recover to a gel state, thus generally requiring noncovalent or dynamic cross-linking. However, a dilemma exists in the design of dynamic hydrogels: hydrogels with fast exchange of cross-links are easier to eject using less force, but lack long-term stability; in contrast, slow exchange of cross-links improves stability, but compromises injectability and thus the ability to protect cells under flow. A new concept to resolve this dilemma using a biocompatible catalyst to modulate the dynamic properties of hydrogels at different time points of application to have both high injectability and high stability is presented. Hyaluronic acid based hydrogels are formed through dynamic covalent hydrazone cross-linking in the presence of a biocompatible benzimidazole-based catalyst. The catalyst accelerates the formation and exchange of hydrazone bonds, enhancing injectability, but rapidly diffuses away from the hydrogel after injection to retard the exchange and improve the long-term stability for cell culture.

We present a new cell culture technology for large-scale mechanobiology studies capable of generating and applying optically controlled uniform compression on single cells in 3D. Mesenchymal stem cells (MSCs) are individually encapsulated inside an optically triggered nanoactuator-alginate hybrid biomaterial using microfluidics, and the encapsulating network isotropically compresses the cell upon activation by light. The favorable biomolecular properties of alginate allow cell culture in vitro up to a week. The mechanically active microgels are capable of generating up to 15% compressive strain and forces reaching 400 nN. As a proof of concept, we demonstrate the use of the mechanically active cell culture system in mechanobiology by subjecting singly encapsulated MSCs to optically generated isotropic compression and monitoring changes in intracellular calcium intensity.