Photolytically-controlled release of dexamethasone [abstract]

Abstract only availableCurrent medical procedures used for repairing bone injuries offer support and stabilization to compromised bones but do not offer a method capable of encouraging bone regeneration. Tissue engineering offers a method of promoting bone regeneration; specifically, we are interested in using tissue engineering to differentiate MSCs into osteoblasts for bone repair. Dexamethasone (dex) has been found to differentiate mesenchymal stem cells (MSCs) into bone-cells, osteoblasts, and we want to control delivery of dex within a tissue engineering scaffold PEG hydrogel. Further, controlled release of dex is important because specific release rates and concentrations (100 nM) are required for the differentiation of MSCs. [1] Externally-controlled release via photolytically labile tethers is beneficial for high-throughput screening of the effect of Dex release on MSCs at differing rates and concentrations. In particular, photolytic release provides control temporally for turning the release on or off with light exposure and tailoring the release rate with light intensity in addition to providing control spatially for location-specific release via photolithography. Here, we used varying light intensities to demonstrate the temporal control granted by using a photolytically labile molecule. Prior to this, we characterized the diffusion of dex within our hydrogel network. Our experiments demonstrated that we could achieve controlled release of dex wih varying light intensity and it should thus be possible to achieve the required 1 week delivery of dex with a low light intensity. On the other hand we observed that using a hydrogel with dex encapsulated would only deliver dex for a matter of hours. For this reason, encapsulation of dex is not a viable option for the differentiation of MSCs. In the future, it is possible that spatial control could be demonstrated by using light to target localized sections of a gel for releasing dex only from those locations.NSF Functional Materials RE

Designing and utilizing synthetic extracellular matrices to probe breast cancer cell activation in response to microenvironment cues

Interactions between breast cancer cells and their microenvironments are essential in tumor growth, metastasis, and recurrence. The tumor stroma undergoes constant structural changes, including degradation, redeposition, and crosslinking of collagens with gradients in matrix stiffness and composition that drive invasion and metastasis. At metastatic sites, similar remodeling events that occur with injury and aging are hypothesized to promote reactivation of dormant tumor cells in recurrence. Approaches are needed for testing hypotheses about pivotal cell-matrix interactions in the progression of breast cancer for identifying key regulators and improving treatment strategies. In this work, we have designed well-defined materials to mimic key aspects of tumor microenvironments toward studying such complex phenomena in vitro. Specifically, we have created synthetic extracellular matrices with well-defined biophysical and biochemical properties that enable three-dimensional (3D) culture of breast cancer cells and niche cells over weeks. A biologically inert polymer, multi-arm poly(ethylene glycol) functionalized with thiols (Mn ~ 20 kDa), was reacted with integrin-binding and cell-degradable peptides decorated with one and two allyloxycarbonyl protecting groups, respectively, by rapid light-triggered thiol-ene polymerization for independent control of matrix density and composition. The elasticity, or ‘stiffness’, of these matrices has been tuned to mimic a variety of soft tissues (Young’s modulus E~0.5-20 kPa), from healthy and cancerous mammary tissues to metastatic site bone marrow and lung tissues. Further, the biochemical content has been tuned with receptor-binding peptides derived from laminin (IKVAV, laminin receptor), collagen ((POG)3POGFOGER(POG)4, α2β1 and α1β1), and fibronectin/vitronectin (RGDS, αVβ3 and α5β1 amongst others) and a crosslinking peptide derived from collagen (GPQG↓IWGQ, degraded MMP-1, -2, and -9 amongst others). We hypothesized that a microenvironment rich in collagen and fibronectin/vitronectin, mimicking aspects of remodeled tissues, would activate breast cancer cells relative to a laminin-rich epithelium-like microenvironment, building upon seminal studies in naturally-derived matrices and in vivo. To test this, we cultured breast cancer cells of different metastatic potential (estrogen receptor positive [ER+, T47Ds] and triple negative [ER-, MDA-MB-231s]) within different matrix densities and compositions. Both cell types exhibited high viability (\u3e 90%), and cell activation in response to different matrix compositions was assayed by examining proliferation (metabolic activity, Ki-67, cell/cluster number and volume) and phenotype (morphology; E-cadherin, vimentin). Increased matrix density decreased elongation of ER- cells and proliferation of both cell types. Increased collagen content increased the proliferation of the ER+ cells and proliferation and elongation of ER- with mass and stellate morphologies, respectively, like observed in naturally-derived matrices. These studies demonstrate a new tool for controlled 3D culture of breast cancer cells relevant for both fundamental and applied research, with on-going investigations incorporating niche cells and triggered matrix changes

Mechanical Properties and Degradation of Chain and Step-Polymerized Photodegradable Hydrogels

The relationship between polymeric hydrogel microstructure and macroscopic properties is of specific interest to the materials science and polymer science communities for the rational design of materials for targeted applications. Specifically, research has focused on elucidating the role of network formation and connectivity on mechanical integrity and degradation behavior. Here, we compared the mechanical properties of chain- and step-polymerized, photodegradable hydrogels. Increased ductility, tensile toughness, and shear strain to yield were observed in step-polymerized hydrogels, as compared to the chain-polymerized gels, indicating that increased homogeneity and network cooperativity in the gel backbone improves mechanical integrity. Furthermore, the ability to degrade the hydrogels in a controlled fashion with light was exploited to explore how hydrogel microstructure influences photodegradation and erosion. Here, the decreased network connectivity at the junction points in the step-polymerized gels resulted in more rapid erosion. Finally, a relationship between the reverse gelation threshold and erosion rate was developed for the general class of photodegradable hydrogels. In all, these studies further elucidate the relationship between hydrogel formation and microarchitecture with macroscale behavior to facilitate the future design of polymer networks and degradable hydrogels, as well as photoresponsive materials such as cell culture templates, drug delivery vehicles, responsive coatings, and anisotropic materials.ISSN:1520-5835ISSN:0024-929

Thiol–ene Click Hydrogels for Therapeutic Delivery

Hydrogels are of growing interest for the delivery of therapeutics to specific sites in the body. For use as a delivery vehicle, hydrophilic precursors are usually laden with bioactive moieties and then directly injected to the site of interest for in situ gel formation and controlled release dictated by precursor design. Hydrogels formed by thiol–ene click reactions are attractive for local controlled release of therapeutics owing to their rapid reaction rate and efficiency under mild aqueous conditions, enabling in situ formation of gels with tunable properties often responsive to environmental cues. Herein, we will review the wide range of applications for thiol–ene hydrogels, from the prolonged release of anti-inflammatory drugs in the spine to the release of protein-based therapeutics in response to cell-secreted enzymes, with a focus on their clinical relevance. We will also provide a brief overview of thiol–ene click chemistry and discuss the available alkene chemistries pertinent to macromolecule functionalization and hydrogel formation. These chemistries include functional groups susceptible to Michael type reactions relevant for injection and radically-mediated reactions for greater temporal control of formation at sites of interest using light. Additionally, mechanisms for the encapsulation and controlled release of therapeutic cargoes are reviewed, including i) tuning the mesh size of the hydrogel initially and temporally for cargo entrapment and release and ii) covalent tethering of the cargo with degradable linkers or affinity binding sequences to mediate release. Finally, myriad thiol–ene hydrogels and their specific applications also are discussed to give a sampling of the current and future utilization of this chemistry for delivery of therapeutics, such as small molecule drugs, peptides, and biologics

Redirecting Valvular Myofibroblasts into Dormant Fibroblasts through Light-mediated Reduction in Substrate Modulus

<div><p>Fibroblasts residing in connective tissues throughout the body are responsible for extracellular matrix (ECM) homeostasis and repair. In response to tissue damage, they activate to become myofibroblasts, which have organized contractile cytoskeletons and produce a myriad of proteins for ECM remodeling. However, persistence of myofibroblasts can lead to fibrosis with excessive collagen deposition and tissue stiffening. Thus, understanding which signals regulate de-activation of myofibroblasts during normal tissue repair is critical. Substrate modulus has recently been shown to regulate fibrogenic properties, proliferation and apoptosis of fibroblasts isolated from different organs. However, few studies track the cellular responses of fibroblasts to dynamic changes in the microenvironmental modulus. Here, we utilized a light-responsive hydrogel system to probe the fate of valvular myofibroblasts when the Young’s modulus of the substrate was reduced from ∼32 kPa, mimicking pre-calcified diseased tissue, to ∼7 kPa, mimicking healthy cardiac valve fibrosa. After softening the substrata, valvular myofibroblasts de-activated with decreases in α-smooth muscle actin (α-SMA) stress fibers and proliferation, indicating a dormant fibroblast state. Gene signatures of myofibroblasts (including α-SMA and connective tissue growth factor (CTGF)) were significantly down-regulated to fibroblast levels within 6 hours of <em>in situ</em> substrate elasticity reduction while a general fibroblast gene vimentin was not changed. Additionally, the de-activated fibroblasts were in a reversible state and could be re-activated to enter cell cycle by growth stimulation and to express fibrogenic genes, such as CTGF, collagen 1A1 and fibronectin 1, in response to TGF-β1. Our data suggest that lowering substrate modulus can serve as a cue to down-regulate the valvular myofibroblast phenotype resulting in a predominantly quiescent fibroblast population. These results provide insight in designing hydrogel substrates with physiologically relevant stiffness to dynamically redirect cell fate <em>in vitro.</em></p> </div

Design of functionalized cyclic peptides through orthogonal click reactions for cell culture and targeting applications

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

Tunable synthetic extracellular matrices to investigate breast cancer response to biophysical and biochemical cues

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

Microgels Formed by Spontaneous Click Chemistries Utilizing Microfluidic Flow Focusing for Cargo Release in Response to Endogenous or Exogenous Stimuli

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