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Khiem A Tran,1,* Michelle Y Cheng,1,* Anupam Mitra,1 Hiromi Ogawa,1 Vivian Y Shi,1 Laura P Olney,1 April M Kloxin,2 Emanual Maverakis1 1Department of Dermatology, University of California, Davis, Sacramento, CA, USA; 2Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA *These authors contributed equally to this work Abstract: The treatment of melanoma has improved markedly over the last several years with the advent of more targeted therapies. Unfortunately, complex compensation mechanisms, such as those of the mitogen-activated protein kinase (MAPK) pathway, have limited the clinical benefit of these treatments. Recently, a better understanding of melanoma resistance mechanisms has given way to intelligently designed multidrug regimes. Herein, we review the extensive pathways of BRAF inhibitor (vemurafenib and dabrafenib) resistance. We also review the advantages of dual therapy, including the addition of an MEK inhibitor (cobimetinib or trametinib), which has proven to increase progression-free survival when compared to BRAF inhibitor monotherapy. Finally, this review touches on future treatment strategies that are being developed for advanced melanoma, including the possibility of triple therapy with immune checkpoint inhibitors and the work on optimizing sequential therapy. Keywords: cobimetinib, trametinib, vemurafenib, dabrafenib, BRAF inhibitor, MAPK pathway

The demand for synthetic soft materials with bioinspired structures continues to grow. Material applications range from in vitro and in vivo tissue mimics to therapeutic delivery systems, where well-defined synthetic building blocks offer precise and reproducible property control. This work examines a synthetic assembling peptide, specifically a multifunctional collagen mimetic peptide (mfCMP) either alone or with reactive macromers, for the creation of responsive hydrogels that capture aspects of soft collagen-rich tissues. We first explored how buffer choice impacts mfCMP hierarchical assembly, in particular, peptide melting temperature, fibril morphology, and ability to form physical hydrogels. Assembly in physiologically relevant buffer resulted in collagen-like fibrillar structures and physically assembled hydrogels with shear-thinning (as indicated through strain-yielding) and self-healing properties. Further, we aimed to create fully synthetic, composite peptide-polymer hydrogels with dynamic responses to various stimuli, inspired by the extracellular matrix (ECM). Specifically, we established mfCMP-poly(ethylene glycol) (PEG) hydrogel compositions that demonstrate increasing non-linear viscoelasticity in response to applied strain as the amount of assembled mfCMP content increases. Furthermore, the thermal responsiveness of mfCMP physical crosslinks was harnessed to manipulate the composite hydrogel mechanical properties in response to changes in temperature. Finally, cells relevant in wound healing, human lung fibroblasts, were encapsulated within these peptide-polymer hydrogels to explore the impact of increased mfCMP, and the resulting changes in viscoelasticity, on cell response. This work establishes mfCMP building blocks as versatile tools for creating hybrid and adaptable systems with applications ranging from injectable shear-thinning materials to responsive interfaces and synthetic ECMs for tissue engineering.

Activation of fibroblasts is pivotal for wound healing; however, persistent activation leads to maladaptive processes and is a hallmark of fibrosis, where disease mechanisms are only partially understood. Human in vitro model systems complement in vivo animal models for both hypothesis testing and drug evaluation to improve the identification of therapeutics relevant to human disease. Despite advances, a challenge remains in understanding the dynamics of human fibroblast responses to complex microenvironment stimuli, motivating the need for more advanced tools to investigate fibrotic mechanisms. This work established approaches for assessing the temporal dynamics of these responses using genetically encoded fluorescent reporters of alpha smooth muscle actin expression, an indicator of fibroblast activation. Specifically, we created a toolset of human lung fibroblast reporter cell lines from different origins (male, female; healthy, idiopathic pulmonary fibrosis) and used three different versions of the reporter with the fluorescent protein modified to exhibit different temporal stabilities, providing temporal resolution of protein expression processes over a range of timescales. Using this toolset, we demonstrated that reporters provide insight into population shifts in response to both mechanical and biochemical cues that are not detectable by traditional end point assessments with differential responses based on cell origin. Furthermore, individual cells can also be tracked over time, with opportunities for comparison to complementary end point measurements. The establishment of this reporter toolset enables dynamic cell investigations that can be translated into more complex synthetic culture environments for elucidating disease mechanisms and evaluating therapeutics for lung fibrosis and other complex biological processes more broadly., Competing Interests: The authors have no conflicts to disclose., (© 2024 Author(s).)

Autoimmune diseases are caused by malfunctions of the immune system and generally impact women at twice the frequency of men. Many of the most serious autoimmune diseases are accompanied by a dysregulation of T-cell phenotype, both regarding the ratio of CD4+ to CD8+ T-cells and proinflammatory versus regulatory phenotypes. Biomaterials, in the form of particles and hydrogels, have shown promise in ameliorating this dysregulation both in vivo and ex vivo. In this review, we explore the role of T-cells in autoimmune diseases, particularly those with high incidence rates in women, and evaluate the promise and efficacy of innovative biomaterial-based approaches for targeting T-cells., Competing Interests: Declaration of Competing Interest The authors declare that no conflict of interest exists., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Hydrogel microparticles ranging from 0.1-100 μm, referred to as microgels, are attractive for biological applications afforded by their injectability and modularity, which allows facile delivery of mixed populations for tailored combinations of therapeutics. Significant efforts have been made to broaden methods for microgel production including via the materials and chemistries by which they are made. Via droplet-based-microfluidics we have established a method for producing click poly-(ethylene)-glycol (PEG)-based microgels with or without chemically crosslinked liposomes (lipo-microgels) through the Michael-type addition reaction between thiol and either vinyl-sulfone or maleimide groups. Unifom spherical microgels and lipo-microgels were generated with sizes of 74 ± 16 μm and 82 ± 25 μm, respectively, suggesting injectability that was further supported by rheological analyses. Super-resolution confocal microscopy was used to further verify the presence of liposomes within the lipo-microgels and determine their distribution. Atomic force microscopy (AFM) was conducted to compare the mechanical properties and network architecture of bulk hydrogels, microgels, and lipo-microgels. Further, encapsulation and release of model cargo (FITC-Dextran 5 kDa) and protein (equine myoglobin) showed sustained release for up to 3 weeks and retention of protein composition and secondary structure, indicating their ability to both protect and release cargos of interest.

Chronic respiratory diseases and infections are among the largest contributors to death globally, many of which still have no cure, including chronic obstructive pulmonary disorder, idiopathic pulmonary fibrosis, and respiratory syncytial virus among others. Pulmonary therapeutics afford untapped potential for treating lung infection and disease through direct delivery to the site of action. However, the ability to innovate new therapeutic paradigms for respiratory diseases will rely on modeling the human lung microenvironment and including key cellular interactions that drive disease. One key feature of the lung microenvironment is the air-liquid interface (ALI). ALI interface modeling techniques, using cell-culture inserts, organoids, microfluidics, and precision lung slices (PCLS), are rapidly developing; however, one major component of these models is lacking-innate immune cell populations. Macrophages, neutrophils, and dendritic cells, among others, represent key lung cell populations, acting as the first responders during lung infection or injury. Innate immune cells respond to and modulate stromal cells and bridge the gap between the innate and adaptive immune system, controlling the bodies response to foreign pathogens and debris. In this article, we review the current state of ALI culture systems with a focus on innate immune cells and suggest ways to build on current models to add complexity and relevant immune cell populations., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023. Published by Elsevier B.V.)

We perform coarse-grained (CG) molecular dynamics (MD) simulations to investigate the self-assembly of collagen-like peptide (CLP) triple helices into fibrillar structures and percolated networks as a function of solvent quality. The focus of this study is on CLP triple helices whose strands are different lengths ( i.e. , heterotrimers), leading to dangling 'sticky ends'. These 'sticky ends' are segments of the CLP strands that have unbonded hydrogen-bonding donor/acceptor sites that drive heterotrimeric CLP triple helices to physically associate with one another, leading to assembly into higher-order structures. We use a validated CG model for CLP in implicit solvent and capture varying solvent quality through changing strength of attraction between CG beads representing the amino acids in the CLP strands. Our CG MD simulations show that, at lower CLP concentrations, CLP heterotrimers assemble into fibrils and, at higher CLP concentrations, into percolated networks. At higher concentrations, decreasing solvent quality causes (i) the formation of heterogeneous network structures with a lower degree of branching at network junctions and (ii) increases in the diameter of network strands and pore sizes. We also observe a nonmonotonic effect of solvent quality on distances between network junctions due to the balance between heterotrimer end-end associations driven by hydrogen bonding and side-side associations driven by worsening solvent quality. Below the percolation threshold, we observe that decreasing solvent quality leads to the formation of fibrils composed of multiple aligned CLP triple helices, while the number of 'sticky ends' governs the spatial extent (radius of gyration) of the assembled fibrils.

Enzymatically degradable peptides are commonly used as linkers within hydrogels for biological applications; however, controlling the degradation of these engineered peptides with different contexts and cell types can prove challenging. In this work, we systematically examined the substitution of d-amino acids (D-AAs) for different l-amino acids in a peptide sequence commonly utilized in enzymatically degradable hydrogels (VPMS↓MRGG) to create peptide linkers with a range of different degradation times, in solution and in hydrogels, and investigated the cytocompatibility of these materials. We found that increasing the number of D-AA substitutions increased the resistance to enzymatic degradation both for free peptide and peptide-linked hydrogels; yet, this trend also was accompanied by increased cytotoxicity in cell culture. This work demonstrates the utility of D-AA-modified peptide sequences to create tunable biomaterials platforms tempered by considerations of cytotoxicity, where careful selection and optimization of different peptide designs is needed for specific biological applications.

Adoptive T-cell therapies (ATCTs) are increasingly important for the treatment of cancer, where patient immune cells are engineered to target and eradicate diseased cells. The biomanufacturing of ATCTs involves a series of time-intensive, lab-scale steps, including isolation, activation, genetic modification, and expansion of a patient's T-cells prior to achieving a final product. Innovative modular technologies are needed to produce cell therapies at improved scale and enhanced efficacy. In this work, well-defined, bioinspired soft materials were integrated within flow-based membrane devices for improving the activation and transduction of T cells. Hydrogel coated membranes (HCM) functionalized with cell-activating antibodies were produced as a tunable biomaterial for the activation of primary human T-cells. T-cell activation utilizing HCMs led to highly proliferative T-cells that expressed a memory phenotype. Further, transduction efficiency was improved by several fold over static conditions by using a tangential flow filtration (TFF) flow-cell, commonly used in the production of protein therapeutics, to transduce T-cells under flow. The combination of HCMs and TFF technology led to increased cell activation, proliferation, and transduction compared to current industrial biomanufacturing processes. The combined power of biomaterials with scalable flow-through transduction techniques provides future opportunities for improving the biomanufacturing of ATCTs., Competing Interests: Conflict of Interest The authors declare that no conflict of interest exists.

Late recurrences of breast cancer are hypothesized to arise from disseminated tumor cells (DTCs) that reactivate after dormancy and occur most frequently with estrogen receptor-positive (ER + ) breast cancer cells (BCCs) in bone marrow (BM). Interactions between the BM niche and BCCs are thought to play a pivotal role in recurrence, and relevant model systems are needed for mechanistic insights and improved treatments. We examined dormant DTCs in vivo and observed DTCs near bone lining cells and exhibiting autophagy. To study underlying cell-cell interactions, we established a well-defined, bioinspired dynamic indirect coculture model of ER + BCCs with BM niche cells, human mesenchymal stem cells (hMSCs) and fetal osteoblasts (hFOBs). hMSCs promoted BCC growth, whereas hFOBs promoted dormancy and autophagy, regulated in part by tumor necrosis factor-α and monocyte chemoattractant protein 1 receptor signaling. This dormancy was reversible by dynamically changing the microenvironment or inhibiting autophagy, presenting further opportunities for mechanistic and targeting studies to prevent late recurrence.

Cells' local mechanical environment can be as important in guiding cellular responses as many well-characterized biochemical cues. Hydrogels that mimic the native extracellular matrix can provide these mechanical cues to encapsulated cells, allowing for the study of their impact on cellular behaviours. Moreover, by harnessing cellular responses to mechanical cues, hydrogels can be used to create tissues in vitro for regenerative medicine applications and for disease modelling. This Primer outlines the importance and challenges of creating hydrogels that mimic the mechanical and biological properties of the native extracellular matrix. The design of hydrogels for mechanobiology studies is discussed, including appropriate choice of cross-linking chemistry and strategies to tailor hydrogel mechanical cues. Techniques for characterizing hydrogels are explained, highlighting methods used to analyze cell behaviour. Example applications for studying fundamental mechanobiological processes and regenerative therapies are provided, along with a discussion of the limitations of hydrogels as mimetics of the native extracellular matrix. The article ends with an outlook for the field, focusing on emerging technologies that will enable new insights into mechanobiology and its role in tissue homeostasis and disease., Competing Interests: Competing interests The authors declare no competing interests.

The pulmonary fibrotic microenvironment is characterized by increased stiffness of lung tissue and enhanced secretion of profibrotic soluble cues contributing to a feedback loop that leads to dysregulated wound healing and lung failure. Pinpointing the individual and tandem effects of profibrotic stimuli in impairing immune cell response remains difficult and is needed for improved therapeutic strategies. We utilized a statistical design of experiment (DOE) to investigate how microenvironment stiffness and interleukin 13 (IL13), a profibrotic soluble factor linked with disease severity, contribute to the impaired macrophage response commonly observed in pulmonary fibrosis. We used engineered bioinspired hydrogels of different stiffness, ranging from healthy to fibrotic lung tissue, and cultured murine alveolar macrophages (MH-S cells) with or without IL13 to quantify cell response and analyze independent and synergistic effects. We found that, while both stiffness and IL13 independently influence macrophage morphology, phenotype, phagocytosis and efferocytosis, these factors work synergistically to exacerbate impaired macrophage phenotype and efferocytosis. These unique findings provide insights into how macrophages in fibrotic conditions are not as effective in clearing debris, contributing to fibrosis initiation/progression, and more broadly inform how underlying drivers of fibrosis modulate immune cell response to facilitate therapeutic strategies.

Protein therapeutics have become increasingly popular for the treatment of a variety of diseases owing to their specificity to targets of interest. However, challenges associated with them have limited their use for a range of ailments, including the limited options available for local controlled delivery. To address this challenge, degradable hydrogel microparticles, or microgels, loaded with model biocargoes were created with tunable release profiles or triggered burst release using chemistries responsive to endogenous or exogeneous stimuli, respectively. Specifically, microfluidic flow-focusing was utilized to form homogenous microgels with different spontaneous click chemistries that afforded degradation either in response to redox environments for sustained cargo release or light for on-demand cargo release. The resulting microgels were an appropriate size to remain localized within tissues upon injection and were easily passed through a needle relevant for injection, providing means for localized delivery. Release of a model biopolymer was observed over the course of several weeks for redox-responsive formulations or triggered for immediate release from the light-responsive formulation. Overall, we demonstrate the ability of microgels to be formulated with different materials chemistries to achieve various therapeutic release modalities, providing new tools for creation of more complex protein release profiles to improve therapeutic regimens.

Collagen-like peptides (CLP) are multifunctional materials garnering a lot of recent interest from the biomaterials community due to their hierarchical assembly and tunable physicochemical properties. In this work, we present a computational study that links the design of CLP heterotrimers to the thermal stability of the triple helix and their self-assembly into fibrillar aggregates and percolated networks. Unlike homotrimeric helices, the CLP heterotrimeric triple helices in this study are made of CLP strands of different chain lengths that result in 'sticky' ends with available hydrogen bonding groups. These 'sticky' ends at one end or both ends of the CLP heterotrimer then facilitate inter-helix hydrogen bonding leading to self-assembly into fibrils (clusters) and percolated networks. We consider the cases of three sticky end lengths - two, four, and six repeat units - present entirely on one end or split between two ends of the CLP heterotrimer. We observe in CLP heterotrimer melting curves generated using coarse grained Langevin dynamics simulations at low CLP concentration that increasing sticky end length results in lower melting temperatures for both one and two sticky ended CLP designs. At higher CLP concentrations, we observe non-monotonic trends in cluster sizes with increasing sticky end length with one sticky end but not for two sticky ends with the same number of available hydrogen bonding groups as the one sticky end; this nonmonotonicity stems from the formation of turn structures stabilized by hydrogen bonds at the single, sticky end for sticky end lengths greater than four repeat units. With increasing CLP concentration, heterotrimers also form percolated networks with increasing sticky end length with a minimum sticky end length of four repeat units required to observe percolation. Overall, this work informs the design of thermoresponsive, peptide-based biomaterials with desired morphologies using strand length and dispersity as a handle for tuning thermal stability and formation of supramolecular structures.

Engineered hydrogels are increasingly used as extracellular matrix (ECM) surrogates for probing cell function in response to ECM remodeling events related to injury or disease (e.g., degradation followed by deposition/crosslinking). Inspired by these events, this work establishes an approach for pseudo-reversible mechanical property modulation in synthetic hydrogels by integrating orthogonal, enzymatically triggered crosslink degradation, and light-triggered photopolymerization stiffening. Hydrogels are formed by a photo-initiated thiol-ene reaction between multiarm polyethylene glycol and a dually enzymatically degradable peptide linker, which incorporates a thrombin-degradable sequence for triggered softening and a matrix metalloproteinase (MMP)-degradable sequence for cell-driven remodeling. Hydrogels are stiffened by photopolymerization using a flexible, MMP-degradable polymer-peptide conjugate and multiarm macromers, increasing the synthetic matrix crosslink density while retaining degradability. Integration of these tools enables sequential softening and stiffening inspired by matrix remodeling events within loose connective tissues (Young's modulus (E) ≈5 to 1.5 to 6 kPa with >3x ΔE). The cytocompatibility and utility of this approach is examined with breast cancer cells, where cell proliferation shows a dependence on the timing of triggered softening. This work provides innovative tools for 3D dynamic property modulation that are synthetically accessible and cell compatible., (© 2022 Wiley-VCH GmbH.)

Recent far-reaching advances in synthetic biology have yielded exciting tools for the creation of new materials. Conversely, advances in the fundamental understanding of soft-condensed matter, polymers and biomaterials offer new avenues to extend the reach of synthetic biology. The broad and exciting range of possible applications have substantial implications to address grand challenges in health, biotechnology and sustainability. Despite the potentially transformative impact that lies at the interface of synthetic biology and biomaterials, the two fields have, so far, progressed mostly separately. This Perspective provides a review of recent key advances in these two fields, and a roadmap for collaboration at the interface between the two communities. We highlight the near-term applications of this interface to the development of hierarchically structured biomaterials, from bioinspired building blocks to 'living' materials that sense and respond based on the reciprocal interactions between materials and embedded cells., (© 2022. Springer Nature Limited.)

The immune system is a complex network of various cellular components that must differentiate between pathogenic bacteria and the commensal bacteria of the human microbiome, where misrecognition is linked to inflammatory disorders. Fragments of bacterial cell wall peptidoglycan bind to pattern recognition receptors within macrophages, leading to immune activation. To study this complex process, a methodology to remodel and label the bacterial cell wall of two different species of bacteria was established using copper (I) catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC). Additionally, an approach for three-dimensional (3D) culture of human macrophages and their invasion with relevant bacteria in a well-defined hydrogel-based synthetic matrix inspired by the microenvironment of the gut was established. Workflows were developed for human monocyte encapsulation and differentiation into macrophages in 3D culture with high viability. Bacteria invaded into macrophages permitted in situ peptidoglycan labeling. Macrophages exhibited biologically-relevant cytokine release in response to bacteria. This molecularly engineered, multi-dimensional bacteria-macrophage co-culture system will prove useful in future studies to observe immunostimulatory, bacterial fragment production and localization in the cell at the carbohydrate level for insights into how the immune system properly senses bacteria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wodzanowski, Caplan, Kloxin and Grimes.)

There is no targeted therapy for triple negative breast cancer (TNBC), which presents an aggressive profile and poor prognosis. Recent studies noticed the feasibility of breast cancer stem cells (BCSCs), a small population responsible for tumor initiation and relapse, to become a novel target for TNBC treatments. However, new cell culture supports need to be standardized since traditional two-dimensional (2D) surfaces do not maintain the stemness state of cells. Hence, three-dimensional (3D) scaffolds represent an alternative to study in vitro cell behavior without inducing cell differentiation. In this work, electrospun polycaprolactone scaffolds were used to enrich BCSC subpopulation of MDA-MB-231 and MDA-MB-468 TNBC cells, confirmed by the upregulation of several stemness markers and the existence of an epithelial-to-mesenchymal transition within 3D culture. Moreover, 3D-cultured cells displayed a shift from MAPK to PI3K/AKT/mTOR signaling pathways, accompanied by an enhanced EGFR and HER2 activation, especially at early cell culture times. Lastly, the fatty acid synthase (FASN), a lipogenic enzyme overexpressed in several carcinomas, was found to be hyperactivated in stemness-enriched samples. Its pharmacological inhibition led to stemness diminishment, overcoming the BCSC expansion achieved in 3D culture. Therefore, FASN may represent a novel target for BCSC niche in TNBC samples., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 Published by Elsevier Ltd.)

Peptides are of continued interest for therapeutic applications, from soluble and immobilized ligands that promote desired binding or uptake to self-assembled supramolecular structures that serve as scaffolds in vitro and in vivo . These applications require efficient and scalable synthetic approaches because of the large amounts of material that often are needed for studies of bulk material properties and their translation. In this work, we establish new methods for the synthesis, purification, and visualization of assembling peptides, with a focus on multifunctional collagen mimetic peptides (mfCMPs) relevant for formation and integration within hydrogel-based biomaterials. First, a methodical approach useful for the microwave-assisted synthesis of assembling peptide sequences prone to deletions was established, beginning with the identification of the deleted residues and their locations and followed by targeted use of dual chemistry couplings for those specific residues. Second, purification techniques that integrate the principles of heating and ion displacement with traditional chromatography and dialysis were implemented to improve separation and isolation of the desired multifunctional peptide product, which contained blocks for thermoresponsiveness and ionic interactions. Third, an approach for fluorescent labeling of these mfCMPs, which is orthogonal to their assembly and their covalent incorporation into a bulk hydrogel material, was established, allowing visualization of the resulting hierarchical fibrillar structures in three dimensions within hydrogels using confocal microscopy. The methods presented in this work allow the production of multifunctional peptides in scalable quantities and with minimal deletions, enabling future studies for better understanding of composition-structure-property relationships and for translating these biomaterials into a range of applications. Although mfCMPs are the focus of this work, the methods demonstrated could prove useful for other assembling peptide systems and for the production of peptides more broadly for therapeutic applications.

The COVID-19 pandemic caused by the global spread of the SARS-CoV-2 virus has led to a staggering number of deaths worldwide and significantly increased burden on healthcare as nations scramble to find mitigation strategies. While significant progress has been made in COVID-19 diagnostics and therapeutics, effective prevention and treatment options remain scarce. Because of the potential for the SARS-CoV-2 infections to cause systemic inflammation and multiple organ failure, it is imperative for the scientific community to evaluate therapeutic options aimed at modulating the causative host immune responses to prevent subsequent systemic complications. Harnessing decades of expertise in the use of natural and synthetic materials for biomedical applications, the biomaterials community has the potential to play an especially instrumental role in developing new strategies or repurposing existing tools to prevent or treat complications resulting from the COVID-19 pathology. Leveraging microparticle- and nanoparticle-based technology, especially in pulmonary delivery, biomaterials have demonstrated the ability to effectively modulate inflammation and may be well-suited for resolving SARS-CoV-2-induced effects. Here, we provide an overview of the SARS-CoV-2 virus infection and highlight current understanding of the host's pulmonary immune response and its contributions to disease severity and systemic inflammation. Comparing to frontline COVID-19 therapeutic options, we highlight the most significant untapped opportunities in immune engineering of the host response using biomaterials and particle technology, which have the potential to improve outcomes for COVID-19 patients, and identify areas needed for future investigations. We hope that this work will prompt preclinical and clinical investigations of promising biomaterials-based treatments to introduce new options for COVID-19 patients.

Assembling peptides allow the creation of structurally complex materials, where amino acid selection influences resulting properties. We present a synergistic approach of experiments and simulations for examining the influence of natural and non-natural amino acid substitutions via incorporation of charged residues and a reactive handle on the thermal stability and assembly of multifunctional collagen mimetic peptides (CMPs). Experimentally, we observed inclusion of charged residues significantly decreased the melting temperature of CMP triple helices with further destabilization upon inclusion of the reactive handle. Atomistic simulations of a single CMP triple helix in explicit water showed increased residue-level and helical structural fluctuations caused by the inclusion of the reactive handle; however, these atomistic simulations cannot be used to predict changes in CMP melting transition. Coarse-grained (CG) simulations of CMPs at experimentally relevant solution conditions, showed, qualitatively, the same trends as experiments in CMP melting transition temperature with CMP design. These simulations show that when charged residues are included electrostatic repulsions significantly destabilize the CMP triple helix and that an additional inclusion of a reactive handle does not significantly change the melting transition. Based on findings from both experiments and simulations, the sequence design was refined for increased CMP triple helix thermal stability, and the reactive handle was utilized for the incorporation of the assembled CMPs within covalently crosslinked hydrogels. Overall, a unique approach was established for predicting stability of CMP triple helices for various sequences prior to synthesis, providing molecular insights for sequence design towards the creation of bulk nanostructured soft biomaterials.

There has been an increased interest in the use of protein therapeutics, especially antibodies, for the treatment of a variety of diseases due to their high specificity to tissues and biological pathways of interest. However, the use of antibodies can be hindered by physical aggregation, degradation, and diffusion when injected in vivo leading to the need for antibody-releasing depots for the controlled and localized delivery within tissues of interest. Here, we investigated photolabile hydrogel chemistries for creating on-demand and tunable antibody release profiles. Innovative, scalable synthetic procedures were established and applied for fabricating hydrogels with nitrobenzyl (NB) and coumarin (CMR) photolabile crosslinks that responded to clinically relevant doses of long-wavelength UV and short-wavelength visible light. This synthetic procedure includes a route to make a CMR linker possessing two functional handles at the same ring position with water-stable bonds. The photocleavage properties of NB and CMR crosslinked hydrogels were characterized, as well as their potential for translational studies by degradation through pig skin, a good human skin mimic. The mechanism of hydrogel degradation, bulk versus surface eroding, was determined to be dependent on the wavelength of light utilized and the molar absorptivity of the different photolabile linkers, providing a facile means for altering protein release upon hydrogel degradation. Further, the encapsulation and on-demand release of a model monoclonal antibody was demonstrated, highlighting the ability to control antibody release from these hydrogels through the application of light while retaining its bioactivity. In particular, the newly designed CMR hydrogels undergo surface erosion-based protein release using visible light, which is more commonly used clinically. Overall, this work establishes scalable syntheses and relevant pairings of formulation-irradiation conditions for designing on-demand and light-responsive material systems that provide controlled, tunable release of bioactive proteins toward addressing barriers to preclinical translation of light-based materials and ultimately improving therapeutic regimens., Competing Interests: The authors declare no conflicts of interest.

Tendon injuries are difficult to heal, in part, because intrinsic tendon healing, which is dominated by scar tissue formation, does not effectively regenerate the native structure and function of healthy tendon. Further, many current treatment strategies also fall short of producing regenerated tendon with the native properties of healthy tendon. There is increasing interest in the use of cell-instructive strategies to limit the intrinsic fibrotic response following injury and improve the regenerative capacity of tendon in vivo . We have established multifunctional, cell-instructive hydrogels for treating injured tendon that afford tunable control over the biomechanical, biochemical, and structural properties of the cell microenvironment. Specifically, we incorporated integrin-binding domains (RGDS) and assembled multifunctional collagen mimetic peptides that enable cell adhesion and elongation of stem cells within synthetic hydrogels of designed biomechanical properties and evaluated these materials using targeted success criteria developed for testing in mechanically demanding environments such as tendon healing. The in vitro and in situ success criteria were determined based on systematic reviews of the most commonly reported outcome measures of hydrogels for tendon repair and established standards for testing of biomaterials. We then showed, using validation experiments, that multifunctional and synthetic hydrogels meet these criteria. Specifically, these hydrogels have mechanical properties comparable to developing tendon; are noncytotoxic both in two-dimensional bolus exposure (hydrogel components) and three-dimensional encapsulation (full hydrogel); are formed, retained, and visualized within tendon defects over time (2-weeks); and provide mechanical support to tendon defects at the time of in situ gel crosslinking. Ultimately, the in vitro and in situ success criteria evaluated in this study were designed for preclinical research to rigorously test the potential to achieve successful tendon repair before in vivo testing and indicate the promise of multifunctional and synthetic hydrogels for continued translation. Impact statement Tendon healing results in a weak scar that forms due to poor cell-mediated repair of the injured tissue. Treatments that tailor the instructions experienced by cells during healing afford opportunities to regenerate the healthy tendon. Engineered cell-instructive cues, including the biomechanical, biochemical, and structural properties of the cell microenvironment, within multifunctional synthetic hydrogels are promising therapeutic strategies for tissue regeneration. In this article, the preclinical efficacy of multifunctional synthetic hydrogels for tendon repair is tested against rigorous in vitro and in situ success criteria. This study indicates the promise for continued preclinical translation of synthetic hydrogels for tissue regeneration.

Synthetic DNA analogues are of great interest for their application in information storage, therapeutics, and nanostructured materials, yet are often limited in scalability. Vinyl sulfonamide click nucleic acids (VS-CNAs) have been developed to overcome this limitation using the highly efficient thiol-Michael 'click' reaction. Utilizing all four nucleobases, sequence-defined click nucleic acids (CNAs) were synthesized using a simple and scalabale solution-phase approach. Employing a polyethylene glycol (PEG) support, synthesis of the CNA sequence, GATTACA, was achieved in high yields. CNA crosslinked hydrogels were assembled using multiarm PEG-CNAs resulting in materials that dynamically respond to temperature, strain, and competitive sequences.

Late recurrences of breast cancer are hypothesized to originate from disseminated tumor cells that re-activate after a long period of dormancy, ≥5 years for estrogen-receptor positive (ER+) tumors. An outstanding question remains as to what the key microenvironment interactions are that regulate this complex process, and well-defined human model systems are needed for probing this. Here, a robust, bioinspired 3D ER+ dormancy culture model is established and utilized to probe the effects of matrix properties for common sites of late recurrence on breast cancer cell dormancy. Formation of dormant micrometastases over several weeks is examined for ER+ cells (T47D, BT474), where the timing of entry into dormancy versus persistent growth depends on matrix composition and cell type. In contrast, triple negative cells (MDA-MB-231), associated with early recurrence, are not observed to undergo long-term dormancy. Bioinformatic analyses quantitatively support an increased "dormancy score" gene signature for ER+ cells (T47D) and reveal differential expression of genes associated with different biological processes based on matrix composition. Further, these analyses support a link between dormancy and autophagy, a potential survival mechanism. This robust model system will allow systematic investigations of other cell-microenvironment interactions in dormancy and evaluation of therapeutics for preventing late recurrence., (© 2020 Wiley-VCH GmbH.)

Wrinkled polymer surfaces find broad applicability; however, the polymer substrates are often limited to poly(dimethylsiloxane) (PDMS), which limits spatial control over wrinkle features and surface chemistry. An approach to surface functionalization of wrinkled elastomer substrates is demonstrated through versatile, multistep thiol-ene click chemistry. The elastomer is formed using a thiol-Michael reaction of tetrathiol with excess diacrylates while wrinkle formation is induced through a second free radical UV polymerization of the acrylates on the surface of the elastomer. Due to oxygen inhibition of the free radical polymerization, pendant acrylates at the surface remain unreacted and are subsequently functionalized with a multi-functional thiol, which can be further reacted through a number of thiol-X 'click' reactions. As a demonstration, these thiol surfaces are further modified to either promote cell adhesion of human mesenchymal stem cells (hMSCs) through coupling with RGDS-containing peptides or surface passivation through reaction with hydrophilic hydroxyl ethyl acrylate moieties. Through engineering a combination of surface chemistry and surface topography, hMSCs exhibited increased spreading and cell density on RGDS-functionalized surfaces and a two-fold increase in cell alignment when cultured on wrinkled substrates. Gradient functionalized surfaces created by tuning the wrinkle wavelength with UV irradiation enabled rapid screening of the effect of topography on the hMSCs. Further, this novel application of click chemistry enables simultaneous tuning of wrinkle topology and surface chemistry towards targeted material applications.

Healthy function of the gut microenvironment is dependent on complex interactions between the bacteria of the microbiome, epithelial and immune (host) cells, and the surrounding tissue. Misregulation of these interactions is implicated in disease. A range of tools have been developed to study these interactions, from mechanistic studies to therapeutic evaluation. In this Digest, we highlight select tools at the cellular and molecular level for probing specific cell-microenvironment interactions. Approaches are overviewed for controlling and probing cell-cell interactions, from transwell and microfluidic devices to engineered bacterial peptidoglycan fragments, and cell-matrix interactions, from three-dimensional scaffolds to chemical handles for in situ modifications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

Photolabile moieties have been utilized in applications ranging from peptide synthesis and controlled protein activation to tunable and dynamic materials. The photochromic properties of nitrobenzyl (NB) based linkers are readily tuned to respond to cytocompatible light doses and are widely utilized in cell culture and other biological applications. While widely utilized, little is known about how the microenvironment, particularly confined aqueous environments (e.g., hydrogels), affects both the mode and rate of cleavage of NB moieties, leading to unpredictable limitations in control over system properties (e.g., rapid hydrolysis or slow photolysis). To address these challenges, we synthesized and characterized the photolysis and hydrolysis of NB moieties containing different labile bonds (i.e., ester, amide, carbonate, or carbamate) that served as labile crosslinks within step-growth hydrogels. We observed that NB ester bond exhibited significant rates of both photolysis and hydrolysis, whereas, importantly, the NB carbamate bond had superior light responsiveness and resistance to hydrolysis within the hydrogel microenvironment. Exploiting this synergy and orthogonality of photolytic and hydrolytic degradation, we designed concentric cylinder hydrogels loaded with different cargoes (e.g., model protein with different fluorophores) for either combinatorial or sequential release, respectively. Overall, this work provides new facile chemical approaches for tuning the degradability of NB linkers and an innovative strategy for the construction of multimodal degradable hydrogels, which can be utilized to guide the design of not only tunable materials platforms but also controlled synthetic protocols or surface modification strategies.

Approaches for the creation of soft materials, particularly hydrogels, with hierarchical structure are of interest in a variety of applications owing to their unique properties. In the context of tissue mimics, hydrogels with multiscale structures more accurately capture the complexities of tissues within the body (e.g., fibrous collagen-rich microenvironments). However, cytocompatible fabrication of such materials with hierarchical structures and independent control of mechanical and biochemical properties remains challenging and is needed for probing and directing cell-microenvironment interactions for three-dimensional (3D) cell encapsulation and culture applications. To address this, we have designed innovative multifunctional assembling peptides: these unique peptides contain a core block that mimics the structure of collagen for achieving relevant melting temperatures; 'sticky' ends to promote assembly of long fibrils; and a biocompatible reactive handle that is orthogonal to assembly to allow the formation of desired multiscale structures and their subsequent rapid, light-triggered integration within covalently crosslinked synthetic hydrogels. Nano- to micro-fibrils were observed to form in physiologically-relevant aqueous solutions, where both underlying peptide chemical structure and assembly conditions were observed to impact the resulting fibril sizes. These assembled structures were 'locked' into place and integrated as linkers within cell-degradable, bioactive hydrogels formed with photoinitiated thiol-ene 'click' chemistry. Hydrogel compositions were identified for achieving robust mechanical properties like those of soft tissues while also retaining higher ordered structures after photopolymerization. The utility of these innovative materials for 3D cell culture was demonstrated with human mesenchymal stem cells, where cell morphologies reminiscent of responses to assembled native collagen were observed now with a fully synthetic material. Using a bottom-up approach, a new materials platform has been established that combines the advantageous properties of covalent and assembling chemistries for the creation of synthetic hydrogels with controllable nanostructure, mechanical properties, and biochemical content.

Therapeutic applications for mesenchymal stem/stromal cells (MSCs) are growing; however, the successful implementation of these therapies requires the development of appropriate MSC delivery systems. Hydrogels are ideally suited to cultivate MSCs but tuning hydrogel properties to match their specific in vivo applications remains a challenge. Thus, further characterization of how hydrogel-based delivery vehicles broadly influence MSC function and fate will help lead to the next generation of more intelligently designed delivery vehicles. To date, few attempts have been made to comprehensively characterize hydrogel impact on the MSC transcriptome. Herein, we have synthesized cell-degradable hydrogels based on bio-inert poly(ethylene glycol) tethered with specific integrin-binding small molecules and have characterized their resulting effect on the MSC transcriptome when compared with 2D cultured and untethered 3D hydrogel cultured MSCs. The 3D culture systems resulted in alterations in the MSC transcriptome, as is evident by the differential expression of genes related to extracellular matrix production, glycosylation, metabolism, signal transduction, gene epigenetic regulation, and development. For example, genes important for osteogenic differentiation were upregulated in 3D hydrogel cultures, and the expression of these genes could be partially suppressed by tethering an integrin-binding RGD peptide within the hydrogel. Highlighting the utility of tunable hydrogels, when applied to ex vivo human wounds the RGD-tethered hydrogel was able to support wound re-epithelialization, possibly due to its ability to increase PDGF expression and decrease IL-6 expression. These results will aid in future hydrogel design for a broad range of applications., (©AlphaMed Press 2019.)

Many cell behaviors are significantly affected by cell culture geometry, though it remains unclear which geometry from two- to three-dimensional (2D to 3D) culture is appropriate for probing a specific cell function and mimicking native microenvironments. Toward addressing this, we established a 2.5D culture geometry, enabling initial cell spreading while reducing polarization to bridge between 2D and 3D geometries, and examined the responses of wound healing cells, human pulmonary fibroblasts, within it. To achieve this, we used engineered biomimetic hydrogels formed by photopolymerization, creating robust layered hydrogels with spread fibroblasts at the interface. We found that fibroblast responses were similar between 2D and 2.5D culture and different from 3D culture, with some underlying differences in mechanotransduction. These studies established the 2.5D cell culture geometry in conjunction with biomimetic synthetic matrices as a useful tool for investigations of fibroblast activation with relevance to the study of other cell functions and types., Competing Interests: Conflicts of interest The authors have no conflicts of interest to declare.

The mechanical properties of synthetic hydrogels traditionally have been controlled with the concentration, molecular weight, or stoichiometry of the macromolecular building blocks used for hydrogel formation. Recently, the rate of formation has been recognized as an important and effective handle for controlling the mechanical properties of these water-swollen polymer networks, owing to differences in network heterogeneity (e.g., defects) that arise based on the rate of gelation. Building upon this, in this work, we investigate a rate-based approach for controlling mechanical properties of hydrogels both initially and temporally with light. Specifically, synthetic hydrogels are formed with visible light-initiated thiol-ene 'click' chemistry (PEG-8-norbornene, dithiol linker, LAP photoinitiator with LED lamp centered at 455 nm), using irradiation conditions to control the rate of formation and the mechanical properties of the resulting hydrogels. Further, defects within these hydrogels were subsequently exploited for temporal modulation of mechanical properties with a secondary cure using low doses of long wavelength UV light (365 nm). The elasticity of the hydrogel, as measured with Young's and shear moduli, was observed to increase with increasing light intensity and concentration of photoinitiator used for hydrogel formation. In situ measurements of end group conversion during hydrogel formation with magic angle spinning (MAS 1 H NMR) correlated with these mechanical properties measurements, suggesting that both dangling end groups and looping contribute to the observed mechanical properties. Dangling end groups provide reactive handles for temporal stiffening of hydrogels with a secondary UV-initiated thiol-ene polymerization, where an increase in Young's modulus by a factor of ~ 2.5x was observed. These studies demonstrate how the rate of photopolymerization can be tuned with irradiation wavelength, intensity, and time to control the properties of synthetic hydrogels, which may prove useful in a variety of applications from coatings to biomaterials for controlled cell culture and regenerative medicine., Competing Interests: Conflicts of interest There are no conflicts to declare.

The extracellular matrix (ECM) is thought to play a critical role in the progression of breast cancer. In this work, we have designed a photopolymerizable, biomimetic synthetic matrix for the controlled, 3D culture of breast cancer cells and, in combination with imaging and bioinformatics tools, utilized this system to investigate the breast cancer cell response to different matrix cues. Specifically, hydrogel-based matrices of different densities and modified with receptor-binding peptides derived from ECM proteins [fibronectin/vitronectin (RGDS), collagen (GFOGER), and laminin (IKVAV)] were synthesized to mimic key aspects of the ECM of different soft tissue sites. To assess the breast cancer cell response, the morphology and growth of breast cancer cells (MDA-MB-231 and T47D) were monitored in three dimensions over time, and differences in their transcriptome were assayed using next generation sequencing. We observed increased growth in response to GFOGER and RGDS, whether individually or in combination with IKVAV, where binding of integrin β1 was key. Importantly, in matrices with GFOGER, increased growth was observed with increasing matrix density for MDA-MB-231s. Further, transcriptomic analyses revealed increased gene expression and enrichment of biological processes associated with cell-matrix interactions, proliferation, and motility in matrices rich in GFOGER relative to IKVAV. In sum, a new approach for investigating breast cancer cell-matrix interactions was established with insights into how microenvironments rich in collagen promote breast cancer growth, a hallmark of disease progression in vivo , with opportunities for future investigations that harness the multidimensional property control afforded by this photopolymerizable system.

Circulating tumor cells (CTCs) play a central role in cancer metastasis and represent a rich source of data for cancer prognostics and therapeutic guidance. Reliable CTC recovery from whole blood therefore promises a less invasive and more sensitive approach to cancer diagnosis and progression tracking. CTCs, however, are exceedingly rare in whole blood, making their quantitative recovery challenging. Several techniques capable of isolating these rare cells have been introduced and validated, yet most suffer from low CTC purity or viability, both of which are essential to develop a clinically viable CTC isolation platform. To address these limitations, we introduce a patterned, immunofunctional, photodegradable poly(ethylene glycol) (PEG) hydrogel capture surface for the isolation and selective release of rare cell populations. Flat and herringbone capture surfaces were successfully patterned via PDMS micromolding and photopolymerization of photolabile PEG hydrogels. Patterned herringbone surfaces, designed to convectively transport cells to the capture surface, exhibited improved capture density relative to flat surfaces for target cell capture from buffer, buffy coat, and whole blood. Uniquely, captured cells were released for collection by degrading the hydrogel capture surface with either bulk or targeted irradiation with cytocompatible doses of long wavelength UV light. Recovered cells remained viable following capture and release and exhibited similar growth rates as untreated control cells. The implementation of molded photodegradable PEG hydrogels as a CTC capture surface provides a micropatternable, cytocompatible platform that imparts the unique ability to recover pure, viable CTC samples by selectively releasing target cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)

As knowledge about the dynamic nature of tissues within the human body has increased, the need for cell culture models that mimic the properties of these dynamic microenvironments has grown. Hydrogels are useful platforms for investigating cellular responses to microenvironment cues in disease and regeneration processes and recently have been designed to contain dynamic bonds to regulate the mechanical and biochemical properties of the matrix in three-dimensional cell culture applications. In this Viewpoint, we highlight recent advances in developing hydrogels with dynamic properties for modeling aspects of human tissues, providing control over the properties of the synthetic matrix on multiple length and time scales, and their application for understanding or directing cell response. We conclude by discussing how orthogonal chemistries can be utilized to design dynamic hydrogel platforms for controlling both the mechanical and biochemical environment, affording opportunities to investigate more complex questions associated with disease progression and tissue regeneration.

The retro Michael-type addition and thiol exchange of thioether succinimide click linkages in response to thiol-containing environments offers a novel strategy for the design of glutathione-sensitive degradable hydrogels for controlled drug delivery. Here we characterize the kinetics and extent of the retro Michael-type addition and thiol exchange with changes in both the p K a of the thiols and the identity of N-substituents of maleimides. A series of N-substituted thioether succinimides were prepared through typical Michael-type addition. Model studies ( 1 H NMR, HPLC) of 4-mercaptophenylacetic acid (MPA, p K a 6.6) conjugated to N-ethyl maleimide (NEM), N-phenyl maleimide (NPM), or N-aminoethyl maleimide (NAEM) and then incubated with glutathione showed half-lives of conversion from 3.1 to 18 h, with extents of conversion from approximately 12% to 90%. The variations in the rates of exchange and hydrolytic ring opening appear to be mediated by resonance effects, electron-withdrawing capacity of the N-substituted moiety, as well as the potential for intramolecular catalytic hydrogen bonding of amine substituents with water (particularly in the case of ring opening). Further model studies of 4-mercaptohydrocinnamic acid (MPP, p K a 7.0) and N-acetyl-l-cysteine (NAC, p K a 9.5) conjugated to selected N-substituted maleimides and then incubated with glutathione showed half-lives of conversion from 3.6 to 258 h, with extents of conversion from approximately 1% to 90%. A higher p K a of the thiol decreased the rate of the exchange reaction and limited the impact of other electronic effects of N-substituents on the extents of conversion. Additional factors affecting the conversion kinetics were studied on NEM conjugates. The kinetics of the retro Michael-type addition and exchange reaction were not hindered by thiol traps of lower p K a , but were retarded in conditions of lower pH. These studies shed light into details of thiol and maleimide design that could be used to tune the rates of degradation of drug and polymer conjugates for a variety of applications.

Synthetic hydrogels have been widely adopted as well-defined matrices for three-dimensional (3D) cell culture, with increasing interest in systems that enable the co-culture of multiple cell types for probing both cell-matrix and cell-cell interactions in studies of tissue regeneration and disease. We hypothesized that the unique dynamic covalent chemistry of self-healing hydrogels could be harnessed for not only the encapsulation and culture of human cells but also the subsequent construction of layered hydrogels for 3D co-cultures. To test this, we formed hydrogels using boronic acid-functionalized polymers and demonstrated their self-healing in the presence of physiologically-relevant cell culture media. Two model human cell lines, MDA-MB-231 breast cancer cells and CCL151 pulmonary fibroblasts, were encapsulated within these dynamic materials, and good viability was observed over time. Finally, self-healing of cut hydrogel 'blocks' laden with these different cell types was used to create layered hydrogels for the generation of a dynamic co-culture system. This work demonstrates the utility of self-healing materials for multi-dimensional cultures and establishes approaches broadly useful for a variety of biological applications.

Synthetic hydrogels with well-defined mechanical properties have become invaluable tools for probing cell response to extracellular cues including matrix stiffness and integrin binding. These synthetic matrices are often decorated with either proteins or integrin-binding peptides to promote cell adhesion and to direct or probe cell behavior. For example, both collagen I-functionalized polyacrylamide and peptide-functionalized poly(ethylene glycol) hydrogels have been instrumental in elucidating the role of the elasticity or 'stiffness' of the matrix in promoting fibroblast activation in wound healing and fibrosis. However, the two methods of promoting integrin binding are not often directly compared in the same system, partly owing to differences in material designs, despite the potential differences in the way cells interact with whole proteins and protein mimetic peptides. We hypothesized that such a comparison could provide insight into the ways integrin binding affects fibroblast activation within commonly utilized in vitro cell culture models, and more broadly, to inform the design of materials to modulate fibroblast activation in studies of wound healing and disease. To enable this comparison, we developed a method to conjugate whole proteins to step-growth poly(ethylene glycol) (PEG) hydrogels and investigated fibroblast response to protein-peptide pairs: fibronectin and PHSRN(G) 10 RGDS or collagen I and (POG) 3 POGFOGER(POG) 4 , which are important in matrix remodeling and relevant to fibroblast activation. With this approach, we observed that human pulmonary fibroblasts adopted a similar morphology on fibronectin and PHSRN(G) 10 RGDS, although with a slight increase in the percentage of alpha smooth muscle actin (αSMA) expressing cells on PHSRN(G) 10 RGDS. Interestingly, we observed that fibroblasts formed activated clusters on the collagen mimic (POG) 3 POGFOGER(POG) 4 while exhibiting less activation on collagen I. This cell activation and clustering is reminiscent of fibroblast foci that are observed in lung fibrosis, suggesting the relevance of these well-defined polymer-peptide hydrogels for investigating fibrosis and decoupling biochemical and biophysical cues.

Controlled, three-dimensional (3D) cell culture systems are of growing interest for both tissue regeneration and disease, including cancer, enabling hypothesis testing about the effects of microenvironment cues on a variety of cellular processes, including aspects of disease progression. In this work, we encapsulate and culture in three dimensions different cancer cell lines in a synthetic extracellular matrix (ECM), using mild and efficient chemistry. Specifically, harnessing the nucleophilic addition of thiols to activated alkynes, we have created hydrogel-based materials with multifunctional poly(ethylene glycol) (PEG) and select biomimetic peptides. These materials have definable, controlled mechanical properties (G' = 4-10 kPa) and enable facile incorporation of pendant peptides for cell adhesion, relevant for mimicking soft tissues, where polymer architecture allows tuning of matrix degradation. These matrices rapidly formed in the presence of sensitive breast cancer cells (MCF-7) for successful encapsulation with high cell viability, greatly improved relative to that observed with the more widely used radically-initiated thiol-ene crosslinking chemistry. Furthermore, controlled matrix degradation by both bulk and local mechanisms, ester hydrolysis of the polymer network and cell-driven enzymatic hydrolysis of cell-degradable peptide, allowed cell proliferation and the formation of cell clusters within these thiol-yne hydrogels. These studies demonstrate the importance of chemistry in ECM mimics and the potential thiol-yne chemistry has as a crosslinking reaction for the encapsulation and culture of cells, including those sensitive to radical crosslinking pathways., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The in situ fabrication of poly(ethylene glycol) diacrylate (PEGDA) hydrogel microstructures within poly(dimethylsiloxane) (PDMS)-based microfluidic networks is a versatile technique that has enabled unique applications in biosensing, medical diagnostics, and the fundamental life sciences. Hydrogel structures have previously been patterned by the lithographic photopolymerization of PEGDA hydrogel forming solutions, a process that is confounded by oxygen-permeable PDMS. Here, we introduce an alternate PEG patterning technique that relies upon the optical sculpting of features by patterned light-induced erosion of photodegradable PEGDA deemed negative projection lithography. We quantitatively compared the hydrogel micropatterning fidelity of negative projection lithography to positive projection lithography, using traditional PEGDA photopolymerization, within PDMS devices. We found that the channel depth, the local oxygen atmosphere, and the UV exposure time dictated the size and resolution of hydrogel features formed using positive projection lithography. In contrast, negative projection lithography was observed to deliver high-resolution functional features with dimensions on the order of single micrometers enabled by its facilely controlled mechanism of feature formation that is insensitive to oxygen. Next, the utility of photodegradable PEGDA was further assessed by encapsulating or conjugating bioactive molecules within photodegradable PEG matrixes to provide a route to the formation of complex and dynamically reconfigurable chemical microenvironments. Finally, we demonstrated that negative projection lithography enabled photopatterning of multilayered microscale objects without the need for precise mask alignment. The described approach for photopatterning high-resolution photolabile hydrogel microstructures directly within PDMS microchannels could enable novel microsystems of increasing complexity and sophistication for a variety of clinical and biological applications., Competing Interests: The authors declare no competing financial interest.

An approach for the design of functionalized cyclic peptides is established for use in 3D cell culture and in cell targeting. Sequential orthogonal click reactions, specifically a photoinitiated thiol-ene and strain promoted azide-alkyne cycloaddition, were utilized for peptide cyclization and conjugation relevant for biomaterial and biomedical applications, respectively.

Induced pluripotent stem cells (iPSCs) are of interest for the study of disease, where these cells can be derived from patients and have the potential to be differentiated into any cell type; however, three-dimensional (3D) culture and differentiation of iPSCs within well-defined synthetic matrices for these applications remains limited. Here, we aimed to establish synthetic cell-degradable hydrogels that allow precise presentation of specific biochemical cues for 3D culture of iPSCs with relevance for hypothesis testing and lineage-specific differentiation. We synthesized poly(ethylene glycol)-(PEG)-peptide-based hydrogels by photoinitiated step growth polymerization and used them to test the hypothesis that the viability of iPSCs within these matrices could be rescued with appropriate biochemical cues inspired by proteins and integrins important for iPSC culture on Matrigel. Specifically, we selected a range of motifs inspired by iPSC binding to Matrigel, including laminin-derived IKVAV and YIGSR, α5β1-binding PHSRNG10RGDS, αvβ5-binding KKQRFRHRNRKG, and RGDS that is known to bind a variety of integrins for generally promoting cell adhesion. YIGSR and PHSRNG10RGDS resulted in the highest iPSC viability, where binding of β1 integrin was key, and these permissive compositions also allowed iPSC differentiation into neural progenitor cells (NPCs) (decreased oct4 expression and increased pax6 expression) in response to soluble factors. The resulting NPCs formed clusters of different sizes in response to each peptide, suggesting that matrix biochemical cues affect iPSC proliferation and clustering in 3D culture. In summary, we have established photopolymerizable synthetic matrices for the encapsulation, culture, and differentiation of iPSCs for studies of cell-matrix interactions and deployment in disease models.

A method of cysteine alkylation using cyclopropenyl ketones is described. Due to the significant release of cyclopropene strain energy, reactions of thiols with cyclopropenyl ketones are both fast and irreversible and give rise to stable conjugate addition adducts. The resulting cyclopropenyl ketones have a low molecular weight and allow for simple attachment of amides via N-hydroxysuccinimide (NHS)-esters. While cyclopropenyl ketones do display slow background reactivity toward water, labeling by thiols is much more rapid. The reaction of a cyclopropenyl ketone with glutathione (GSH) proceeds with a rate of 595 M -1 s -1 in PBS at pH 7.4, which is considerably faster than α-halocarbonyl labeling reagents, and competitive with maleimide/thiol couplings. The method has been demonstrated in protein conjugation, and an arylthiolate conjugate was shown to be stable upon prolonged incubation in either GSH or human plasma. Finally, cyclopropenyl ketones were used to create PEG-based hydrogels that are stable to prolonged incubation in a reducing environment.

Cells interact with and remodel their microenvironment, degrading large extracellular matrix (ECM) proteins (e.g., fibronectin, collagens) and secreting new ECM proteins and small soluble factors (e.g., growth factors, cytokines). Synthetic mimics of the ECM have been developed as controlled cell culture platforms for use in both fundamental and applied studies. However, how cells broadly remodel these initially well-defined matrices remains poorly understood and difficult to probe. In this work, we have established methods for widely examining both large and small proteins that are secreted by cells within synthetic matrices. Specifically, human mesenchymal stem cells (hMSCs), a model primary cell type, were cultured within well-defined poly(ethylene glycol) (PEG)-peptide hydrogels, and these cell-matrix constructs were decellularized and degraded for subsequent isolation and analysis of deposited proteins. Shotgun proteomics using liquid chromatography and mass spectrometry identified a variety of proteins, including the large ECM proteins fibronectin and collagen VI. Immunostaining and confocal imaging confirmed these results and provided visualization of protein organization within the synthetic matrices. Additionally, culture medium was collected from the encapsulated hMSCs, and a Luminex assay was performed to identify secreted soluble factors, including vascular endothelial growth factor (VEGF), endothelial growth factor (EGF), basic fibroblast growth factor (FGF-2), interleukin 8 (IL-8), and tumor necrosis factor alpha (TNF-α). Together, these methods provide a unique approach for studying dynamic reciprocity between cells and synthetic microenvironments and have the potential to provide new biological insights into cell responses during three-dimensional (3D) controlled cell culture., Competing Interests: The authors declare no competing financial interest.

Injectable delivery systems that respond to biologically relevant stimuli present an attractive strategy for tailorable drug release. Here, the design and synthesis of unique polymers are reported for the creation of hydrogels that are formed in situ and degrade in response to clinically relevant endogenous and exogenous stimuli, specifically reducing microenvironments and externally applied light. Hydrogels are formed with polyethylene glycol and heparin-based polymers using a Michael-type addition reaction. The resulting hydrogels are investigated for the local controlled release of low molecular weight proteins (e.g., growth factors and cytokines), which are of interest for regulating various cellular functions and fates in vivo yet remain difficult to deliver. Incorporation of reduction-sensitive linkages and light-degradable linkages affords significant changes in the release profiles of fibroblast growth factor-2 (FGF-2) in the presence of the reducing agent glutathione or light, respectively. The bioactivity of the released FGF-2 is comparable to pristine FGF-2, indicating the ability of these hydrogels to retain the bioactivity of cargo molecules during encapsulation and release. Further, in vivo studies demonstrate degradation-mediated release of FGF-2. Overall, our studies demonstrate the potential of these unique stimuli-responsive chemistries for controlling the local release of low molecular weight proteins in response to clinically relevant stimuli., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Hydrogel-based depots are of growing interest for release of biopharmaceuticals; however, a priori selection of hydrogel compositions that will retain proteins of interest and provide desired release profiles remains elusive. Toward addressing this, in this work, we have established a new tool for the facile assessment of protein release from hydrogels and applied it to evaluate the effectiveness of mesh size estimations on predicting protein retention or release. Poly(ethylene glycol) (PEG)-based hydrogel depots were formed by photoinitiated step growth polymerization of four-arm PEG functionalized with norbornene (PEG-norbornene, 4% w/w to 20% w/w, M n ∼ 5 to 20 kDa) and different dithiol cross-linkers (PEG M n ∼ 1.5 kDa or enzymatically degradable peptide), creating well-defined, robust materials with a range of mesh sizes estimated with Flory-Rehner or rubber elasticity theory (∼5 to 15 nm). A cocktail of different model proteins was released from compositions of interest, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to facilely and quantitatively analyze temporal release profiles. Mesh size was predictive of retention of relatively large proteins and release of relatively small proteins. Proteins with diameters comparable to the mesh size, which is often the case for growth factors, were released by hindered diffusion and required experimental assessment of retention and release. With this knowledge, hydrogels were designed for the controlled release of a therapeutically relevant growth factor, PDGF-BB.

Hydrogels are facile architectures for the controlled presentation of proteins with far-reaching applications, from fundamental biological studies in three-dimensional culture to new regenerative medicine and therapeutic delivery strategies. Here, we demonstrate a versatile approach for spatially-defined presentation of engineered proteins within hydrogels through i) immobilization using bio-orthogonal strain-promoted alkyne-azide click chemistry and ii) dynamic protease-driven protein release using exogenously applied enzyme. Model fluorescent proteins were expressed using nonsense codon replacement to incorporate azide-containing unnatural amino acids in a site-specific manner toward maintaining protein activity: here, cyan fluorescent protein (AzCFP), mCherry fluorescent protein (AzmCh), and mCh decorated with a thrombin cut-site. (AzTMBmCh). Eight-arm poly(ethylene glycol) (PEG) was modified with dibenzylcyclooctyne (DBCO) groups and reacted with azide functionalized PEG in aqueous solution for rapid formation of hydrogels. Azide functionalized full-length fluorescent proteins were successfully incorporated into the hydrogel network by reaction with PEG-DBCO prior to gel formation. Temporal release and removal of select proteins (AzTMBmCh) was triggered with the application of thrombin and monitored in real-time with confocal microscopy, providing a responsive handle for controlling matrix properties. Hydrogels with regions of different protein compositions were created using a layering technique with thicknesses of hundreds of micrometers, affording opportunities for the creation of complex geometries on size scales relevant for controlling cellular microenvironments., Statement of Significance: Controlling protein presentation within biomaterials is important for modulating interactions with biological systems. For example, native tissues are composed of subunits with different matrix compositions (proteins, stiffness) that dynamically interact with cells, influencing function and fate. Toward mimicking such temporally-regulated and spatially-defined microenvironments, we utilize bio-orthogonal click chemistry and protein engineering to create hydrogels with distinct regions of proteins and modify them over time. Through nonsense codon replacement, we site-specifically functionalize large proteins with i) azides for covalent conjugation and ii) an enzymatic cleavage site for user-defined release from hydrogels. Our results exemplify not only the ability to create unique bio-functionalized hydrogels with controlled mechanical properties, but also the potential for creating interesting interfaces for cell culture and tissue engineering applications., (Copyright © 2017. Published by Elsevier Ltd.)

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol-ene and thiol-yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol-X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol-ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.