Project description:Polymer-nanoparticle (PNP) hydrogels are a class of nanocomposite materials showing potential as injectable platforms for biomedical applications. Their design is limited by incomplete knowledge of how the binding motif impacts the viscoelastic properties of the material and is generally constrained to non-responsive supramolecular interactions. Expanding the scope of available interactions and advancing the understanding of how defined interactions influence network formation would accelerate PNP hydrogel design. To address this gap in the design of PNP hydrogels, the study designs and investigates a tunable platform based on beta-cyclodextrin (βCD) host-guest cross-links between functionalized polymers and nanoparticles. A host-functionalized polymer (βCD hyaluronic acid) and guest harboring block co-polymer (poly(ethylene glycol)-b-poly(lactic acid)) NPs are synthesized. The presence and accessibility for binding of the host and guest moieties are characterized via isothermal titration calorimetry. PNP hydrogels with varying concentrations of functionalized polymer and NPs reveal a limited window of concentrations for gelation. It is hypothesized that network formation is governed by the capacity of polymer chains to effectively bridge NPs, which is related to the host-guest ratios present in the system. Further, photo-responsive guests are incorporated to engineer photoreversible gelation of PNP hydrogels via exposure to specific wavelengths of light.
Project description:EM has long been the main technique for imaging cell structures with nanometer resolution but has lagged behind light microscopy in the crucial ability to make specific molecules stand out. Here we introduce click-EM, a labeling technique for correlative light microscopy and EM imaging of nonprotein biomolecules. In this approach, metabolic labeling substrates containing bioorthogonal functional groups are provided to cells for incorporation into biopolymers by endogenous biosynthetic machinery. The unique chemical functionality of these analogs is exploited for selective attachment of singlet oxygen-generating fluorescent dyes via bioorthogonal 'click chemistry' ligations. Illumination of dye-labeled structures generates singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product that is readily imaged by EM. We describe the application of click-EM in imaging metabolically tagged DNA, RNA and lipids in cultured cells and neurons and highlight its use in tracking peptidoglycan synthesis in the Gram-positive bacterium Listeria monocytogenes.
Project description:Injectable gelatin hydrogels formed with bioorthogonal click chemistry (ClickGel) are cell-responsive ECM mimics for in vitro and in vivo biomaterials applications. Gelatin polymers with pendant norbornene (GelN) or tetrazine (GelT) groups can quickly and spontaneously crosslink upon mixing, allowing for high viability of encapsulated cells, establishment of 3D elongated cell morphologies, and biodegradation when injected in vivo.
Project description:Hydrogels cross-linked by inverse electron demand Diels-Alder (iEDDA) click chemistry are increasingly used in biomedical applications. With a few exceptions in naturally derived and chemically modified macromers, iEDDA click hydrogels exhibit long-term hydrolytic stability, and no synthetic iEDDA click hydrogels can undergo accelerated and tunable hydrolytic degradation. We have previously reported a novel method for synthesizing norbornene (NB)-functionalized multiarm poly(ethylene glycol) (PEG), where carbic anhydride (CA) was used to replace 5-norbornene-2-carboxylic acid. The new PEGNBCA-based thiol-norbornene hydrogels exhibited unexpected fast yet highly tunable hydrolytic degradation. In this contribution, we leveraged the new PEGNBCA macromer for forming iEDDA click hydrogels with [methyl]tetrazine ([m]Tz)-modified macromers, leading to the first group of synthetic iEDDA click hydrogels with highly tunable hydrolytic degradation kinetics. We further exploited Tz and mTz dual conjugation to achieve tunable hydrolytic degradation with an in vitro degradation time ranging from 2 weeks to 3 months. Finally, we demonstrated the excellent in vitro cytocompatibility and in vivo biocompatibility of the new injectable PEGNBCA-based iEDDA click cross-linked hydrogels.
Project description:The inverse electron demand Diels-Alder (iEDDA) reactions are highly efficient click chemistry increasingly utilized in bioconjugation, live cell labeling, and the synthesis and modification of biomaterials. iEDDA click reactions have also been used to cross-link tetrazine (Tz) and norbornene (NB) modified macromers [e.g., multiarm poly(ethylene glycol) or PEG]. In these hydrogels, Tz-NB adducts exhibit stable supramolecular interactions with a high hydrolytic stability. Toward engineering a new class of PEG-based click hydrogels with highly adaptable properties, we previously reported a new group of NB-derivatized PEG macromers via reacting hydroxyl-terminated PEG with carbic anhydride (CA). In this work, we show that hydrogels cross-linked by PEGNBCA or its derivatives exhibited fast and tunable hydrolytic degradation. Here, we show that PEGNBCA (either mono- or octafunctional) and its dopamine or tyramine conjugated derivatives (i.e., PEGNB-D and PEGNB-T) readily cross-link with 4-arm PEG-Tz to form a novel class of multifunctional iEDDA click hydrogels. Through modularly adjusting the macromers with unstable and stable iEDDA click-induced supramolecular interactions (iEDDA-CSI), we achieved highly tunable degradation, with full degradation in less than 2 weeks to over two months. We also show that secondary enzymatic reactions could dynamically stiffen these hydrogels. These hydrogels could also be spatiotemporally photopatterned through visible light-initiated photochemistry. Finally, the iEDDA-CSI hydrogels post ester hydrolysis displayed shear-thinning and self-healing properties, enabling injectable delivery.
Project description:Oxime Click chemistry was used to form hydrogels that support cell adhesion. Eight-armed aminooxy poly(ethylene glycol) (PEG) was mixed with glutaraldehyde to form oxime-linked hydrogels. The mechanical properties, gelation kinetics, and water swelling ratios were studied and found to be tunable. It was also shown that gels containing the integrin ligand arginine-glycine-aspartic acid (RGD) supported mesenchymal stem cell (MSC) incorporation. High cell viability and proliferation of the encapsulated cells demonstrated biocompatibility of the material.
Project description:Poly(ethylene glycol) (PEG)-based synthetic hydrogels based on Michael-type addition reaction have been widely used for cell culture and tissue engineering. However, recent studies showed that these types of hydrogels were not homogenous as expected since micro domains generated due to the fast reaction kinetics. Here, we demonstrated a new kind of method to prepare homogenous poly(ethylene glycol) hydrogels based on Michael-type addition using the side chain amine-contained short peptides. By introducing such a kind of short peptides, the homogeneity of crosslinking and mechanical property of the hydrogels has been also significantly enhanced. The compressive mechanical and recovery properties of the homogeneous hydrogels prepared in the presence of side chain amine-contained short peptides were more reliable than those of inhomogeneous hydrogels while the excellent biocompatibility remained unchanged. Furthermore, the reaction rate and gelation kinetics of maleimide- and thiol-terminated PEG were proved to be significantly slowed down in the presence of the side chain amine-contained short peptides, thus leading to the improved homogeneity of the hydrogels. We anticipate that this new method can be widely applied to hydrogel preparation and modification based on Michael-type addition gelation.
Project description:The Diels-Alder reaction is recognized to generate highly selective and regiospecific cycloadducts. In this study, we carried out a rheological and kinetic study of N-furfuryl chitosan hydrogels based on the Diels-Alder click reaction with different poly(ethylene)glycol-maleimide derivatives in dilute aqueous acidic solutions. It was possible to prepare clear and transparent hydrogels with excellent mechanical properties. Applying the Winter and Chambon criterion the gel times were estimated at different temperatures, and the activation energy was calculated. The higher the temperature of gelation, the higher the reaction rate. The crosslinking density and the elastic properties seem to be controlled by the diffusion of the polymer segments, rather than by the kinetics of the reaction. An increase in the concentration of any of the two functional groups is accompanied by a higher crosslinking density regardless maleimide:furan molar ratio. The hydrogel showed an improvement in their mechanical properties as the temperature increases up to 70 °C. Above that, there is a drop in G' values indicating that there is a process opposing to the Diels-Alder reaction, most likely the retro-Diels-Alder.
Project description:Understanding the spatiotemporal effects of surface topographies and modulated stiffness and anisotropic stresses of hydrogels on cell growth remains a biophysical challenge. Here we introduce the photolithographic patterning or two-photon laser scanning confocal microscopy patterning of a series of o-nitrobenzylphosphate ester nucleic acid-based polyacrylamide hydrogel films generating periodically-spaced circular patterned domains surrounded by continuous hydrogel matrices. The patterning processes lead to guided modulated stiffness differences between the patterned domains and the surrounding hydrogel matrices, and to the selective functionalization of sub-regions of the films with nucleic acid anchoring tethers. HeLa cells are deposited on the circularly-shaped domains functionalized with the MUC-1 aptamers. Initiation of the hybridization chain reaction by nucleic acid tethers associated with the continuous hydrogel matrix results in stress-induced ordered orthogonal shape-changes on the patterned domains, leading to ordered shapes of cell aggregates bound to the patterns.