Evaluation of the Higher Order Structure of Biotherapeutics Embedded in Hydrogels for Bioprinting and Drug Release.

Domenico Rizzo,Anna Pérez-Ràfols,Linda Cerofolini,Fabio Baroni,Claudio Luchinat,Stefano Giuntini,Marco Fragai,Enrico Ravera

doi:10.1021/acs.analchem.1c01850

Abstract

Biocompatible hydrogels for tissue regeneration/replacement and drug release with specific architectures can be obtained by three-dimensional bioprinting techniques. The preservation of the higher order structure of the proteins embedded in the hydrogels as drugs or modulators is critical for their biological activity. Solution nuclear magnetic resonance (NMR) experiments are currently used to investigate the higher order structure of biotherapeutics in comparability, similarity, and stability studies. However, the size of pores in the gel, protein–matrix interactions, and the size of the embedded proteins often prevent the use of this methodology. The recent advancements of solid-state NMR allow for the comparison of the higher order structure of the matrix-embedded and free isotopically enriched proteins, allowing for the evaluation of the functionality of the material in several steps of hydrogel development. Moreover, the structural information at atomic detail on the matrix–protein interactions paves the way for a structure-based design of these biomaterials.

Highlights

The continuous development of new biocompatible materials is opening new frontiers in medicine and new biotechnological opportunities
Article the use of solid-state Nuclear magnetic resonance (NMR) has been described to characterize noncrystalline large protein assemblies,[44−50] biomaterials,[51,52] bioinspired silica matrix embedding enzymes,[53−58] conjugated proteins,[59−62] protein-grafted nanoparticles,[63] and vaccines.[64−66] Here, we prove that solid-state NMR provides detailed information on the preservation of the higher order structure (HOS) of proteins embedded into two popular matrices used for 3D bioprinting
■ RESULTS AND DISCUSSION Analysis of the Preservation of the HOS of the Proteins Encapsulated in the Hyaluronic Acid Hydrogel by solid-state NMR (SSNMR)

Summary

■ INTRODUCTION

The continuous development of new biocompatible materials is opening new frontiers in medicine and new biotechnological opportunities. 1C,D) are of high quality and comparable, for the number of cross-peaks detected, with those of rehydrated freeze-dried proteins For both proteins embedded in the hyaluronic acid matrix, the matching of the resonances of the 2D-NMR spectral fingerprints with those of their own reference allows us to assess the preservation of the HOS after encapsulation in the matrix. The analyses of the chemical shift perturbation (CSP) of the NCA spectra of the proteins embedded in the hyaluronic acid hydrogels, with respect to the NCA of the corresponding rehydrated freeze-dried references, are reported in Figures 2 and 3. It was possible to acquire a 2D 13C-13C correlation spectrum at 20 kHz, which allowed us to assess the folding state of the protein in the hydrogel and, after comparison with that acquired for the rehydrated freeze-dried reference (Figure S4), confirm the preservation of the HOS after encapsulation. The observation that hydrophobic and polar neutral amino acids on the protein surface experience the largest effects provides a way to design possible chemical modifications of the matrix in order to tune the protein−matrix interactions and the properties of the resulting biomaterial.[78−82]

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES