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- ItemGelation kinetics of thiol-methylsulfone (MS) hydrogel formulations for 3D cell culture(Washington, D.C. : American Chemical Society, 2022) de Miguel-Jiménez, Adrián; Ebeling, Bastian; Paez, Julieta I.; Fink-Straube, Claudia; Pearson, Samuel; del Campo, AranzazuCrosslinking chemistries that allow hydrogel formation within minutes are essential to achieve homogeneous networks and cell distributions in 3D cell culture. Thiol-methylsulfone (MS) crosslinking chemistry offers minutes-scale gelation under near-physiological conditions showing many desirable attributes for 3D cell encapsulation. Here we investigate the gelation kinetics and mechanical properties of PEG-based hydrogels formed by thiol-tetrazole methylsulfone (TzMS) crosslinking as a function of buffer, crosslinker structure, and degree of TzMS functionalization. Appropriate buffer selection ensured constant pH throughout crosslinking. The formulation containing cell adhesive ligand RGD and enzymatically-degradable peptide VPM gelled in ca. 4 min at pH 7.5, and stiffness could be increased from hundreds of Pascals to > 1 kPa by using excess VPM. The gelation times and stiffnesses for these hydrogels are highly suitable for 3D cell encapsulations, and pave the way for reliable 3D cell culture workflows in pipetting robots.
- ItemA photoactivatable α5β1-specific integrin ligand(ChemRxiv, 2018) Vakkeel, Roshna; Aleeza, Farrukh; del Campo, AranzazuIn order to study how dynamic changes of α5β1 integrin engagement affect cellular behaviour, photoactivatable derivatives of α5β1 specific ligands are presented in this article. The presence of the photoremovable protecting group (PRPG) introduced at a relevant position for integrin recognition, temporally inhibits ligand bioactivity. Light exposure at cell-compatible dose efficiently cleaves the PRPG and restores functionality. Selective cell response (attachment, spreading, migration) to the activated ligand on the surface is achieved upon controlled exposure. Spatial and temporal control of the cellular response is demonstrated, including the possibility to in situ activation. Photoactivatable integrin-selective ligands in model microenvironments will allow the study of cellular behavior in response to changes in the activation of individual integrins as consequence of dynamic variations of matrix composition.
- ItemPhotoactivatable Hsp47: A tool to control and regulate collagen secretion & assembly(ChemRxiv, 2018) Khan, Essak; Sankaran, Shrikrishnan; Paez, Julieta; Muth, Christina; Han, Mitchell; del Campo, AránzazuHsp47 is a chaperone protein with a fundamental role in the folding, stability and intracellular transport of procollagen triple helices. A light-responsive Hsp47 recombinant protein, engineered to control in situ the production and assembly of cellular collagen is here demonstrated. This novel light-driven tool enables unprecedented fundamental studies of collagen biosynthesis and associated diseases.
- ItemRedox-triggerable Luciferin-Bioinspired Hydrogels as Injectable and Cell-encapsulating Matrices(Washington, D.C. : American Chemical Society, 2022) Jin, Minye; Gläser, Alisa; Paez, Julieta I.Over the past few decades there has been a great interest in developing smart hydrogels that are stimuli-responsive, due to their ability to respond to variations caused by external stimuli. These materials are exploited for biomedical applications such as biosensors, injectable scaffolds, drug delivery and tissue engineering. Recently, our group reported firefly-inspired hydrogel matrices for 3D cell culture. This platform exhibited certain advantages like rapid gelation rate and tunability of mechanical and biological properties. However, this firstly reported system did not allow for fine control of the gelation onset because the crosslinking reaction started as soon as the two precursors were mixed. Moreover, one of its precursors demonstrated poor storage stability in aqueous solution. These limitations restrict its application as injectable matrices. In this article, we endow the luciferin-inspired hydrogels with redox-triggering capability, to overcome the limitations of the previous system and to widen its application range. We achieve this goal by introducing protected macromers as hydrogel polymeric precursors that can be activated in the presence of a mild reductant, to trigger gel formation in situ with high degree of control. We demonstrate that the regulation of intrinsic (e.g., structure of protecting group, reductant type) and extrinsic (e.g., pH, temperature) parameters of the triggering reaction can be used to modulate key materials properties. This novel upgraded redox-triggerable system enables precise control over gelation onset and kinetics, thus facilitating its utilization as injectable hydrogel without negatively impacting its cytocompatibility. Our findings expand the current toolkit of chemically-based stimuli-responsive hydrogels.
- ItemPossibilities and Limitations of Photoactivatable Cytochalasin D for the Spatiotemporal Regulation of Actin Dynamics(Washington, D.C. : American Chemical Society, 2020) Nair, Roshna V.; Zhao, Shifang; Terriac, Emmanuel; Lautenschläger, Franziska; Hetmanski, Joseph H.R.; Caswell, Patrick T.; del Campo, AranzazuThe study of the actin cytoskeleton and related cellular processes requires tools to specifically interfere with actin dynamics in living cell cultures, ideally with spatiotemporal control and compatible with real time imaging. A phototriggerable derivative of the actin disruptor Cytochalasin D (CytoD) is described and tested here. It includes a nitroveratryloxycarbonyl (Nvoc) photoremovable protecting group (PPG) at the hydroxyl group at C7 of CytoD. The attachment of the PPG renders Nvoc-CytoD temporarily inactive, and enables light-dosed delivery of the active drug CytoD to living cells. This article presents the full structural and physicochemical characterization, the toxicity analysis. It is complemented with biological tests to show the time scales (seconds) and spatial resolution (cellular level) achievable with a UV source in a regular microscopy setup