Since its conception by Sharpless in 20011
, the concept of click chemistry has been rapidly adopted in many disciplines, perhaps most notably in material science, with annual publication numbers continuing to increase exponentially2–5
. The click philosophy idealizes reactions that enable researchers to link covalently two reactants in a straightforward, modular, high-yielding manner. The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is frequently billed as the quintessential example in meeting these criteria. Though each of the click reactions has a variety of desirable properties, their true benefit lies in the orthogonality of these reactions with respect to many common reactive groups (e.g.
, amines, alcohols, acids)6
. Reaction orthogonality enables independent control over multiple functional groups in a single system and opens the door for the synthesis of materials with ever-increasing complexity for an ever-expanding list of applications.
There is also a growing interest in chemical reactions that can be performed in the presence of live cells6
. These reactions must proceed mildly in aqueous medium and a defined chemical environment (5% CO2
and atmospheric O2
levels), under regulated pH (7.4), temperature (37°C), osmolarity (~300 mOsM), and involve non-toxic reactive moieties and (by)products. The requirement of reactions that proceed under physiological conditions is a stringent constraint and presents severe limitations on reaction selection. In particular, there are even fewer chemistries that proceed in a specific manner, while limiting side reactions with the plethora of functional groups that are found in biological systems. These elite reactions are considered bio-orthogonal and are a necessity for probing chemically and directing biological function.
In many instances, the ideal cytocompatible reaction would not only be selected by its bio-orthogonality, but also by its capacity to be controlled in both time and space. In this regard, photochemical reactions are widely regarded for their spatiotemporal control, where the reaction of interest is defined by when and where the light is delivered to the system7
. Photolithographic techniques, where masked light is projected directly onto a sample, enable photoreactions to be confined to specific regions within a sample as defined by a 2D mask pattern, while focused laser light (either single- or multi-photon) provides full 3D control over where a specific reaction occurs within the volume of a material. While the effects of attenuation and scattering must be carefully taken into consideration, light-based chemistries have become a powerful tool for material synthesis and spatial modification, owing to their ease of implementation and readily available inexpensive light sources, and have become indispensible in formation and subsequent modification of biomaterials.
To date, biocompatible light-based chemistries have enabled control in space and time of either
the gel degradation or gel chemistry of synthetic cell culture systems. By introducing chemical functionalities in user-defined patterns within the material, cell spreading and migration have been explicitly controlled in 3D8–13
. Alternatively, cell outgrowth and stem cell fate have been directed by altering the gel’s structural properties14–18
. Nevertheless, independent control over both
the material’s physical and chemical makeup in 3D, along with in time, allow dynamic tailoring of a cell’s microenvironment, and has not been demonstrated. Such 4D control of material properties would be tremendously advantageous in a number of biomaterial applications, including 3D cell culture, stem cell expansion, cancer metastasis, and tissue regeneration. Of further importance and novelty, the ability for the experimenter to control gel properties at any point in space and time enables opportunities for unique experiments, such as the ability to introduce dynamically a cell ligand or allow cell-cell interactions at specified locations. These programmable cell culture niches facilitate the ability to perform newfound experiments and answer questions about the dynamic exchange of information between a cell and its niche. In this work, we present one such system where multiple wavelengths of light are utilized to control independently the functionality and architecture of a hydrogel network formed via
a copper-free alkyne-azide reaction (). Each of the reactions is cytocompatible, and both photoconjugation and photocleavage reactions were used to spatiotemporally regulate materials properties, including the presentation of integrin-binding motifs and network erosion through cleavage of crosslinking moieties. This platform allows gel parameters to be tuned in real-time, and results demonstrate how spatiotemporal regulation of material properties can be used to direct the function of embedded cells.
Synthesis, photocoupling, and photodegradation for tuning chemical and physical properties of click-based hydrogels