RESUMO
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.
Assuntos
Ciclopropanos/química , Cisteína/química , Cetonas/química , Alquilação , Glutationa/química , Humanos , Hidrogéis/síntese química , Polietilenoglicóis , Coloração e Rotulagem , Compostos de Sulfidrila/química , Fatores de TempoRESUMO
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.
RESUMO
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.
Assuntos
Neoplasias da Mama , Técnicas de Cultura de Células/métodos , Modelos Biológicos , Receptores de Estrogênio/metabolismo , Microambiente Tumoral/fisiologia , Autofagia , Neoplasias da Mama/química , Neoplasias da Mama/metabolismo , Neoplasias da Mama/fisiopatologia , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Feminino , Humanos , Biologia SintéticaRESUMO
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 ß1 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.
RESUMO
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.
RESUMO
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.
RESUMO
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.
Assuntos
Química Click/métodos , Hidrogéis/química , Luz , Peptídeos/química , Fotoquímica/métodos , Materiais Biocompatíveis , Técnicas de Cultura de Células , Sistemas de Liberação de Medicamentos , Humanos , Células-Tronco Mesenquimais/citologia , Polietilenoglicóis/química , Polimerização , Compostos de Sulfidrila/química , Engenharia TecidualRESUMO
Thiol-ene 'click' chemistries have been widely used in biomaterials applications, including drug delivery, tissue engineering, and controlled cell culture, owing to their rapid, cytocompatible, and often orthogonal reactivity. In particular, hydrogel-based biomaterials formed by photoinitiated thiol-ene reactions afford spatiotemporal control over the biochemical and biomechanical properties of the network for creating synthetic materials that mimic the extracellular matrix or enable controlled drug release. However, the use of charged peptides functionalized with cysteines, which can form disulfides prior to reaction, and vinyl monomers that require multistep syntheses and contain ester bonds, may lead to undesired inhomogeneity or degradation under cell culture conditions. Here, we designed a thiol-ene hydrogel formed by the reaction of allyloxycarbonyl-functionalized peptides and thiol-functionalized poly(ethylene glycol). Hydrogels were polymerized by free radical initiation under cytocompatible doses of long wavelength ultraviolet light in the presence of water-soluble photoinitiators (lithium acylphosphinate, LAP, and 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone, Irgacure 2959). Mechanical properties of these hydrogels were controlled by varying the monomer concentration to mimic a range of soft tissue environments, and hydrogel stability in cell culture medium was observed over weeks. Patterns of biochemical cues were created within the hydrogels post-formation and confirmed through the incorporation of fluorescently-labeled peptides and Ellman's assay to detect free thiols. Human mesenchymal stem cells remained viable after encapsulation and subsequent photopatterning, demonstrating the utility of the monomers and hydrogels for three-dimensional cell culture. This facile approach enables the formation and characterization of hydrogels with well-defined, spatially-specific properties and expands the suite of monomers available for three-dimensional cell culture and other biological applications.
RESUMO
Wound healing results from complex signaling between cells and their environment in response to injury. Fibroblasts residing within the extracellular matrix (ECM) of various connective tissues are critical for matrix synthesis and repair. Upon injury or chronic insult, these cells activate into wound-healing cells, called myofibroblasts, and repair the damaged tissue through enzyme and protein secretion. However, misregulation and persistence of myofibroblasts can lead to uncontrolled accumulation of matrix proteins, tissue stiffening, and ultimately disease. Extracellular cues are important regulators of fibroblast activation and have been implicated in their persistence. Hydrogel-based culture models have emerged as useful tools to examine fibroblast response to ECM cues presented during these complex processes. In this Mini-Review, we will provide an overview of these model systems, which are built upon naturally-derived or synthetic materials, and mimic relevant biophysical and biochemical properties of the native ECM with different levels of control. Additionally, we will discuss the application of these hydrogel-based systems for the examination of fibroblast function and fate, including adhesion, migration, and activation, as well as approaches for mimicking both static and temporal aspects of extracellular environments. Specifically, we will highlight hydrogels that have been used to investigate the effects of matrix rigidity, protein binding, and cytokine signaling on fibroblast activation. Last, we will describe future directions for the design of hydrogels to develop improved synthetic models that mimic the complex extracellular environment.