LETTERS PUBLISHED ONLINE: 21 JUNE 2009 | DOI: 10.1038/NMAT2473 Sequential click reactions for synthesizing and patterning three-dimensional cell microenvironments Cole A. DeForest1, Brian D. Polizzotti1,2 and Kristi S. Anseth1,2* Click chemistry provides extremely selective and orthogonal chemistry has been exploited in the labelling of biomolecules, it has reactions that proceed with high efficiency and under a variety not yet been used for biomaterial formation. of mild conditions, the most common example being the More recently, the radical-mediated addition of a thiol to an copper(I)-catalysed reaction of azides with alkynes1,2. While alkene known as the thiol–ene reaction has gained attention as the versatility of click reactions has been broadly exploited3–5, an emerging click reaction18. In addition to being bio-orthogonal a major limitation is the intrinsic toxicity of the synthetic and biocompatible, the reaction is advantageous in that it is schemes and the inability to translate these approaches readily initiated with light, ultimately affording spatial and temporal into biological applications. This manuscript introduces a control over where the reaction occurs19. This reaction has been robust synthetic strategy where macromolecular precursors used to create two-dimensional surface gradients of biomolecules20 react through a copper-free click chemistry6, allowing for as well complex materials21. the direct encapsulation of cells within click hydrogels In alignment with the evolution of click chemistry, the combined for the first time. Subsequently, an orthogonal thiol–ene utilization of multiple orthogonal reactions presents the oppor- photocoupling chemistry is introduced that enables patterning tunity to fabricate multifunctional and tunable materials without of biological functionalities within the gel in real time and with sacrificing synthetic simplicity or efficiency. While materials with micrometre-scale resolution. This material system enables highly defined structures have applications in microelectronics, us to tailor independently the biophysical and biochemical membrane technology, and fuel cells, one increasingly important properties of the cell culture microenvironments in situ. This area of research is in developing biomaterial platforms that enable synthetic approach uniquely allows for the direct fabrication of researchers to culture and study cells in three dimensions22. Though biologically functionalized gels with ideal structures that can be initial material development has proven successful in permitting photopatterned, and all in the presence of cells. cell growth, a growing topic of interest is the development of An emerging paradigm in organic synthesis is a focus on bioactive materials that promote and detect specific cell func- highly selective and orthogonal reactions that proceed with high tion through spatially presented biochemical and biomechanical efficiency and under a variety of mild conditions. A growing cues23. Ultimately, a platform offering such versatility would be number of these reactions are grouped under the term `click of particular note to those interested in well-defined niches for chemistry', and have been used to produce a catalogue of functional three-dimensional (3D) cell culture and understanding the role synthetic molecules and subsequent materials1,4. Characteristics of of biomechanical versus biochemical signals in cell function, as modular click reactions include (1) high yields with fast kinetics, well as regenerating tissue structures24. Appropriately developed (2) regiospecificity and stereospecificity, (3) insensitivity to oxygen click chemistry can provide this versatility, enabling the fabrica- or water and (4) mild reaction conditions, solventless or in water. tion of increasingly complex 3D culture constructs using just a While the versatility of click reactions has been broadly few simple reactions. exploited in many fields including drug discovery7,8, material Here, a hydrogel platform is introduced that uses two orthog- science9–11, and bioconjugation3,12,13, a major limitation is the onal click chemistries; one for hydrogel formation and another intrinsic toxicity of the synthetic schemes and the inability to for biochemical patterning within the preformed material. The translate these approaches to biological applications. Though the modular aspect of these reactions allows for independent control 1,3-dipolar Huisgen cycloaddition between azides and alkynes2 of the network structure and chemistry, and facile incorporation is often seen as the quintessential click reaction, the crucial of biological epitopes. Network formation is accomplished using copper catalyst precludes its use with biological systems14,15. a recently developed Cu-free variant to the traditional click re- This drawback, however, was recently circumvented through the action, the Huisgen cycloaddition, between an azide (–N3) and development of novel cyclooctyne moieties whose ring strain an alkyne (–C≡C–) to form a triazole6. This method uses a and electron-withdrawing fluorine substituents give rise to an di-fluorinated cyclooctyne moiety (DIFO3), whose ring strain and activated alkyne. This molecule has been shown to react quickly electron-withdrawing fluorine substituents promote the T3 C 2U with azides in the absence of a metal catalyst, enabling the use dipolar cycloaddition with azides without the use of a catalyst25 of traditional click chemistry in living systems6,16. Specifically, (Fig. 1a). This reaction has been carried out under physiological azide-labelled cell-surface glycans were reacted with fluorescently conditions in the presence of living cells with no reported toxicity17. labelled cyclooctynes in vivo to enable the visualization of dynamic Beyond this bioconjugation approach for cell labelling, multifunc- subcellular development within zebra-fish embryos17. Though this tional macromolecular monomers were synthesized to create ideal 1Department of Chemical and Biological Engineering; 2Howard Hughes Medical Institute, University of Colorado, UCB Box 424 Boulder, Colorado 80309-0424, USA. *e-mail: [email protected]. NATURE MATERIALS j VOL 8 j AUGUST 2009 j www.nature.com/naturematerials 659 © 2009 Macmillan Publishers Limited. All rights reserved. LETTERS NATURE MATERIALS DOI: 10.1038/NMAT2473 a O O O O O O O O O O H H H H H H H H H C NCHC NCHC NCHC NCHC NCHC NCHC NCHC NCHC NCHC NH2 O O O O O O O CH H CH CH H CH CH CH CH H H H H H H 2 3 CH2 2 2 2 2 H3C C NCHC NCHC NCHC N CH C NCHC NCHC N CH2 CH2 CH CH3 CH2 CH2 CH2 CH2 CH CH CH CH H H 2 2 2 2 C O CH3 CH3 C O CH2 CH2 CH2 CH CH CH CH 2 2 2 2 NH2 NH2 NH NH CH2 CH2 CH2 CH2 CH2 C NH C NH NH CH NH NH CH Enzymatically degradable O C 2 2 NH2 NH2 NH C NH C NH NH peptide sequence H2C C O NH2 NH2 C O F CH 2 O Photoreactive group = F F CH2 F HC for patterning CH2 + N3 Aqueous O 37 °C O O O O O N3 = O N3 O N3 Poly(ethylene glycol) Tetra-azide Mol. wt.: ~10,000 R2 N R2 Aqueous R N+ + F 1 N¬ F 37 °C F F N N R1 N Ideal network 3D gel b 105 c Gel point (~5 min) 103 Complete gelation (~1 h) 103 101 101 Moduli (Pa) 10¬1 10¬1 Moduli (Pa) 10¬3 0 200 400 600 Time (s) 100 µm 10¬3 0 1,000 2,000 3,000 Time (s) Figure 1 j Cytocompatible-click-hydrogel formation reaction and kinetics. a, Click-functionalized macromolecular precursors undergo the T3C2U Huisgen cycloaddition to form a 3D ideal network hydrogel through a step-growth polymerization mechanism. b, Rheology can be used to monitor dynamic network formation and indicates gelation within minutes and complete reaction occurring in less than 1 h at 37 ◦Cfor a 13.5 wt% monomer solution. G0 is shown as closed circles, whereas G00 are open circles. c, A live/dead stain at 24 h of 3T3s encapsulated within this material indicates a predominantly viable population (live cells are shown in green, whereas dead cells are shown in red). The image represents a 200 µm confocal projection. network structures with minimal defects and local heterogeneities. metalloproteinase cleavable sequence (GPQG # ILGQ) is selected, Specifically, multifunctional azides and activated alkynes were re- so that cells can actively remodel their surroundings through acted in a one-to-one fashion to yield end-linked polymer gels, secreted enzymes26. Cells encapsulated in hydrogels containing an under reaction conditions that enable cell encapsulation and result enzymatically degradable crosslinker sequence spread and migrate in gels with initially uniform material properties. throughout the material, with dramatically increased viability A four-arm poly(ethylene glycol) (PEG) tetra-azide was reacted compared with non-degradable alternatives27,28. with bis(DIFO3) di-functionalized polypeptide in an aqueous Hydrogels were formed using a 13.5 wt% total macromer environment at 37 ◦C (schematic shown in Fig. 1a). The choice solution containing a 1:1 ratio of alkyne to azide functionalities. of PEG enables us to tailor readily the biophysical properties Ultimately, this gel composition affords a high water content, of the gel, while eliminating non-specific interactions that often elasticity similar to many tissue matrices, and the ability to result when proteins adsorb to materials. Biological functionality image cells in three dimensions. Dynamic time sweep rheological can be readily introduced into the hydrogel backbone by the experiments were conducted to monitor network evolution during choice of the crosslinking peptide sequence. Here, a matrix this step polymerization (Fig. 1b). The crossover point, an estimate 660 NATURE MATERIALS j VOL 8 j AUGUST 2009 j www.nature.com/naturematerials © 2009 Macmillan Publishers Limited. All rights reserved. NATURE MATERIALS DOI: 10.1038/NMAT2473 LETTERS ab0.7 c HS S Constant intensity, varied exposure R1 R R 2 1 0.6 Constant exposure, varied intensity 0.5 0.4 R S S 0.3 2 R1 R1 0.2 Concentration (mM) Concentration 0.1 100 µm 0 R2 02468101214 UV light S R1 + HS R1 Dosage (J cm¬2) d e 100 µm 50 µm Figure 2 j Cytocompatible, biochemical patterning within preformed click hydrogels.