Introduction: Hydrogel microspheres are attractive biomaterials for the delivery of cells and protein therapeutics and as building blocks for the assembly of 3D scaffolds. While a variety of methods for fabricating hydrogel microspheres have been described in the literature, free radical chain polymerization remains the most commonly used chemistry. In recent years, however, click chemistry reactions (i.e., reactions that are fast, efficient, specific, and mild) have emerged as powerful tools that offer superior control over the synthesis of bioactive materials. We are particularly interested in photoinitiated thiol-norbornene click chemistry, which is exceptionally fast and efficient, and bio-orthogonal tetrazine-norbornene click chemistry, which is an efficient and mild radical-free chemistry. Importantly, these two click reactions are complementary and can be performed sequentially because both utilize norbornene functional groups. The goal of this project was to leverage these two click reactions to synthesize protein-functionalized poly(ethylene glycol) (PEG) based hydrogel microspheres with tunable bioactive properties.
Materials and Methods: PEG-based hydrogel microparticles were fabricated using an immersed electrospraying apparatus and thiol-norbornene click chemistry. Briefly, aqueous 6 wt.% PEG-tetra-norbornene (20kDa) was combined with a di-thiol crosslinker (dithiothreitol; enzymatically cleavable peptide) at a [thiol]:[norbornene] ratio of 0.75, and 2mM lithium acylphosphinate photoinitiator. This solution was extruded at varying flow rates (100-500µL/min) and voltages (3-7kV) from a 22-gauge blunt needle immersed in mineral oil and located 15 mm from a grounded electrode. The collected droplets were then photopolymerized using 365 nm light and characterized by light microspcopy. Subsequently, the unreacted norbornenes were leveraged for protein conjugation with tetrazine click chemistry. Fluorescein-ovalbumin and alkaline phosphatase (ALP) modified with a 1,2,4,5-tetrazine were used as a model proteins. Ovalbumin conjugation was verified by fluorescence microscopy and flow cytometry. ALP bioactivity was characterized based on p-nitrophenyl phosphate conversion and alizarin red staining after incubation in calcium glycerophosphate solution.
Results and Discussion: Immersed electrospraying and thiol-ene click chemistry enabled efficient hydrogel microsphere polymerization, and microsphere size was tunable between 30-300µm based on the flow rate and applied voltage. Moreover, the stoichiometric excess of norbornene functional groups in the thiol-ene polymerization allowed for protein conjugation with tetrazine click chemistry. Conjugation of fluorescein-ovalbumin was confirmed based on fluorescence. ALP bioactivity tests revealed that the amount of tetrazine-functionalized ALP (Tz-ALP) added correlated positively with microsphere bioactivity, whereas non-functionalized control ALP (NF-ALP) resulted in minimal bioactivity (Fig. 1). These results indicate that protein bioactivity was preserved and tunable.
Fig. 1. Bioactive hydrogel microspheres via click chemistry.
Conclusion: This work demonstrates the use of sequential click reactions to synthesize protein-functionalized PEG hydrogel microspheres with tunable size and bioactivity. This approach could have broad utility for applications in drug delivery and tissue engineering.