A Modular Approach to Sensitized Two-Photon Patterning of Photodegradable Hydrogels.
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ABSTRACT: Photodegradable hydrogels have emerged as useful platforms for research on cell function, tissue engineering, and cell delivery as their physical and chemical properties can be dynamically controlled by the use of light. The photo-induced degradation of such hydrogel systems is commonly based on the integration of photolabile o-nitrobenzyl derivatives to the hydrogel backbone, because such linkers can be cleaved by means of one- and two-photon absorption. Herein we describe a cytocompatible click-based hydrogel containing o-nitrobenzyl ester linkages between a hyaluronic acid backbone, which is photodegradable in the presence of cells. It is demonstrated for the first time that by using a cyclic benzylidene ketone-based small molecule as photosensitizer the efficiency of the two-photon degradation process can be improved significantly. Biocompatibility of both the improved two-photon micropatterning process as well as the hydrogel itself is confirmed by cell culture studies.
Project description:Photodegradable hydrogels have emerged as a powerful material platform for studying and directing cell behaviors, as well as for delivering drugs. The premise of this technique is to use a cytocompatible light source to cleave linkers within a hydrogel, thus causing reduction of matrix stiffness or liberation of matrix-tethered biomolecules in a spatial-temporally controlled manner. The most commonly used photodegradable units are molecules containing nitrobenzyl moieties that absorb light in the ultraviolet (UV) to lower visible wavelengths (~280 to 450 nm). Because photodegradable linkers and hydrogels reported in the literature thus far are all sensitive to UV light, highly efficient UV-mediated photopolymerizations are less likely to be used as the method to prepare these hydrogels. As a result, currently available photodegradable hydrogels are formed by redox-mediated radical polymerizations, emulsion polymerizations, Michael-type addition reactions, or orthogonal click chemistries. Here, we report the first photodegradable poly(ethylene glycol)-based hydrogel system prepared by step-growth photopolymerization. The model photolabile peptide cross-linkers, synthesized by conventional solid phase peptide synthesis, contained terminal cysteines for step-growth thiol-ene photo-click reactions and a UV-sensitive 2-nitrophenylalanine residue in the peptide backbone for photo-cleavage. Photolysis of this peptide was achieved through adjusting UV light exposure time and intensity. Photopolymerization of photodegradable hydrogels containing photolabile peptide cross-linkers was made possible via a highly efficient visible light-mediated thiol-ene photo-click reaction using a non-cleavage type photoinitiator eosin-Y. Rapid gelation was confirmed by in situ photo-rheometry. Flood UV irradiation at controlled wavelength and intensity was used to demonstrate the photodegradability of these photopolymerized hydrogels.
Project description:Artificial 3-dimensional (3D) cell culture systems, which mimic the extracellular matrix (ECM), hold great potential as models to study cellular processes under controlled conditions. The natural ECM is a 3D structure composed of a fibrous hydrogel that provides both mechanical and biochemical cues to instruct cell behavior. Here we present an ECM-mimicking genetically engineered protein-based hydrogel as a 3D cell culture system that combines several key features: (1) Mild and straightforward encapsulation meters (1) ease of ut I am not so sure.encapsulation of the cells, without the need of an external crosslinker. (2) Supramolecular assembly resulting in a fibrous architecture that recapitulates some of the unique mechanical characteristics of the ECM, i.e. strain-stiffening and self-healing behavior. (3) A modular approach allowing controlled incorporation of the biochemical cue density (integrin binding RGD domains). We tested the gels by encapsulating MG-63 osteoblastic cells and found that encapsulated cells not only respond to higher RGD density, but also to overall gel concentration. Cells in 1% and 2% (weight fraction) protein gels showed spreading and proliferation, provided a relative RGD density of at least 50%. In contrast, in 4% gels very little spreading and proliferation occurred, even for a relative RGD density of 100%. The independent control over both mechanical and biochemical cues obtained in this modular approach renders our hydrogels suitable to study cellular responses under highly defined conditions.
Project description:Hydrogel scaffolds are commonly used as 3D carriers for cells because their properties can be tailored to match natural extracellular matrix. Hydrogels may be used in tissue engineering and regenerative medicine to deliver therapeutic cells to injured or diseased tissue through controlled degradation. Hydrolysis and enzymolysis are the two most common mechanisms employed for hydrogel degradation, but neither allows sequential or staged release of cells. In contrast, photodegradation allows external real-time spatial and temporal control over hydrogel degradation, and allows for staged and sequential release of cells. We synthesized and characterized a series of macromers incorporating photodegradbale ortho-nitrobenzyl (o-NB) groups in the macromer backbone. We formed hydrogels from these macromers via redox polymerization and quantified the apparent rate constants of degradation (kapp) of each via photorheology at 370 nm, 10 mW/cm(2). Decreasing the number of aryl ethers on the o-NB group increases kapp, and changing the functionality from primary to seconday at the benzylic site dramatically increases kapp. Human mesenchymal stem cells (hMSCs) survive encapsulation in the hydrogels (90% viability postencapsulation). By exploiting the differences in reactivity of two different o-NB linkers, we quantitatively demonstrate the biased release of one stem cell population (green-fluoroescent protein expressing hMSCs) over another (red-fluorescent protein expressing hMSCs).
Project description:We report a strategy to create photodegradable poly(ethylene glycol)-based hydrogels through rapid polymerization of cytocompatible macromers for remote manipulation of gel properties in situ. Postgelation control of the gel properties was demonstrated to introduce temporal changes, creation of arbitrarily shaped features, and on-demand pendant functionality release. Channels photodegraded within a hydrogel containing encapsulated cells allow cell migration. Temporal variation of the biochemical gel composition was used to influence chondrogenic differentiation of encapsulated stem cells. Photodegradable gels that allow real-time manipulation of material properties or chemistry provide dynamic environments with the scope to answer fundamental questions about material regulation of live cell function and may affect an array of applications from design of drug delivery vehicles to tissue engineering systems.
Project description:Silicon nanocrystals of the average diameter of 5 nm, functionalized with 4,7-di(2-thienyl)-2,1,3-benzothiadiazole chromophores (TBT) and dodecyl chains, exhibit near-infrared emission upon one-photon (1P) excitation at 515 nm and two-photon (2P) excitation at 960 nm. By using TBT chromophores as an antenna we were able to enhance both 1P and 2P absorption cross-sections of the silicon nanocrystals to more efficiently excite their long-lived luminescence. These results chart a path to two-photon-excitable imaging probes with long-lived oxygen-independent luminescence - a rare combination of properties that should allow for a substantial increase in imaging contrast.
Project description:Cells may be captured and released using a photodegradable hydrogel (photogel) functionalized with antibodies. Photogel substrates were used to first isolate human CD4 or CD8 T-cells from a heterogeneous cell suspension and then to release desired cells or groups of cells by UV-induced photodegradation. Flow cytometry analysis of the retrieved cells revealed approximately 95% purity of CD4 and CD8 T-cells, suggesting that this substrate had excellent specificity. To demonstrate the possibility of sorting cells according to their function, photogel substrates that were functionalized with anti-CD4 and anti-TNF-? antibodies were prepared. Single cells captured and stimulated on such substrates were identified by the fluorescence "halo" after immunofluorescent staining and could be retrieved by site-specific exposure to UV light through a microscope objective. Overall, it was demonstrated that functional photodegradable hydrogels enable the capture, analysis, and sorting of live cells.
Project description: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, 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, degradable hydrogels, as well as photoresponsive materials as cell culture templates, drug delivery vehicles, responsive coatings, and anisotropic materials.
Project description:Cell sorting is an essential and efficient experimental tool for the isolation and characterization of target cells. A three-dimensional environment is crucial in determining cell behavior and cell fate in biological analysis. Herein, we have applied photodegradable hydrogels to optical cell separation from a 3D environment using a computer-controlled light irradiation system. The hydrogel is composed of photocleavable tetra-arm polyethylene glycol and gelatin, which optimized cytocompatibility to adjust a composition of crosslinker and gelatin. Local light irradiation could degrade the hydrogel corresponding to the micropattern image designed on a laptop; minimum resolution of photodegradation was estimated at 20 µm. Light irradiation separated an encapsulated fluorescent microbead without any contamination of neighbor beads, even at multiple targets. Upon selective separation of target cells in the hydrogels, the separated cells have grown on another dish, resulting in pure culture. Cell encapsulation, light irradiation and degradation products exhibited negligible cytotoxicity in overall process.
Project description:This paper describes the generation of "click-crosslinkable" and "photodegaradable" gelatin hydrogels from the reaction between dibenzocycloctyl-terminated photoclevable tetra-arm polyethylene glycol and azide-modified gelatin. The hydrogels were formed in 30 min through the click-crosslinking reaction. The micropatterned features in the hydrogels were created by micropatterned light irradiation; the minimum resolution of micropatterning was 10-μm widths for line patterns and 20-μm diameters for circle patterns. Cells were successfully encapsulated in the hydrogels without any loss of viability across a wide concentration range of crosslinker. In contrast, an activated-ester-type photocleavable crosslinker, which we previously used to prepare photodegradable gelatin hydrogels, induced a decrease in cell viability at crosslinker concentrations greater than 1.8 mM. We also observed morphology alteration and better growth of cancer cells in the click-crosslinked photodegradable gelatin hydrogels that included matrigel than in the absence of matrigel. We also demonstrated micropatterning of the hydrogels encapsulating cells and optical cell separation. Both of the cells that remained in the non-irradiated area and the cells collected from the irradiated area maintained their viability.