The components of cellular microenvironments, especially the extracellular matrix (ECM), strongly regulate biological processes through biochemical and mechanical signaling. For this, hydrogels have been widely used to build artificial niches mimicking the native ECM. Strain-promoted azide-alkyne cycloaddition (SPAAC) is a biorthogonal reaction between strained cyclic ring-containing alkynes and azides, which proceeds under mild, copper-free, biocompatible conditions. Here, we developed an amphiphile SPAAC-clickable alginate derivative capable of forming ionic hydrogels with hydrophobic microdomains, which can be further functionalized in-situ and in the presence of cells. This system provides topographical cues to cells within a true 3D microenvironment and may sequester molecules with hydrophobic moieties while allowing on-demand dynamic switch of matrix properties.
Ultra-pure alginate was functionalized with a cyclooctyne (BCN-amine) by carbodiimide chemistry. Modification degrees of alkyne-alginates (ALK) were assessed by 1H-NMR. ALK derivatives were characterized by contact angle measurements, hydrophobic probes, scanning electron cryomicroscopy, and viscometry. ALK hydrogels (acellular and cell-laden) were prepared by ionic crosslinking with Ca2+ . Mechanical analysis was performed by microindentation and rheometry. SPAAC conjugation with azide-functionalized compounds was performed at 37ºC, in pre-gel solutions (0.9% w/v NaCl) or pre-formed hydrogels (culture medium). Grafting kinetics and efficiency were estimated using fluorescent azido-tags. Clickable hydrogels laden with mesenchymal stem cells (MSC) were analyzed in metabolic activity and morphology, ECM protein expression by immunostaining, and gene expression for 14-d.
ALK with varying modification degrees was successfully produced. Increased modification produced ALK derivatives with less hydrophilicity, stronger interactions with hydrophobic probes, and increased viscosity in aqueous solutions. At higher modification degrees, ALK derivatives showed the ability to spontaneously establish concentration-dependent associations between polymer chains. Ionic ALK hydrogels with denser microstructures within a sparser 3D network were produced, showing spatial heterogeneity in stiffness, as expected. These regions not only added topographical features to the otherwise smooth bulk hydrogel but also provided binding regions for sequestering compounds with hydrophobic sites, such as proteins, as verified using an extrinsic hydrophobic fluorescent probe. In-situ and on-demand multi-functionalization was confirmed by performing consecutive SPAAC conjugations. Reactions proceeded rapidly (< 30 min) under physiological conditions (i.e., in culture medium at 37ºC). In MSC-laden ALK hydrogels, cells remained viable and metabolically active throughout culture time. Cell spreading and extracellular fibronectin expression were detected at the microdomain regions only, which worked as topographical harbors for cell anchoring.
By taking advantage of the intrinsic hydrophobicity of cyclooctyne groups, which can be conjugated with azido-conjugated compounds via SPAAC, we successfully formulated hydrogels that present topographical cues to cells in a true 3D microenvironment, while also allowing dynamic, on-demand, (bio)functionalized in the presence of cells.
References:  Kim, E, et al., Chem Sci 10(34) (2019) 7835-7851.  DeForest, CA, et al., Nature Materials 8(8) (2009) 659-664.  Jain, E, et al., ACS Applied Bio Materials 4(2) (2021) 1229-1237.  Torres, AL, et al., Biomaterials 228 (2020) 119554.
Acknowledgment: Portuguese Foundation for Science and Technology (FCT) for project EndoSWITCH (PTDC/BTM-ORG/5154/2020), fellowship SFRH/BD/129855/2017, and contract IF/00296/2015