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

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.

Epitope Mapping and Binding Assessment by Solid-State NMR Provide a Way for the Development of Biologics under the Quality by Design Paradigm

“…A few years ago, a pioneering work by the group of Lewandowski reported the solid-state NMR characterization of a precipitated macromolecular complex between the first immunoglobulin binding domain of streptococcal protein G (GB1) and a full-length antibody . GB1 is a 6 kDa protein that is extensively used as a standard in solid-state NMR, and is reported to bind strongly to the crystallizable region fragment and weakly to the antigen-binding fragment of human immunoglobulin G. These results and previous studies on noncrystalline systems suggest that also very large macromolecular systems involving fusion-derived biologics can be characterized by solid-state NMR spectroscopy. − One of the advantages of the noncrystalline samples, obtained by sedimentation or equivalently by rehydrating freeze-dried proteins, is the absence of crystalline (ordered) packing . Indeed, the shift perturbations due to the contacts among the different protein molecules are averaged over several poses with no energetic preferences and the hydration state of the biomolecules is closer to that present in solution. , Therefore, a rehydrated freeze-dried material corresponds to an extremely concentrated solution of the protein, which is intrinsically comparable, for the scope of chemical shift mapping, to the diluted sample used for acquiring solution spectra …”

Combining Solid‐State NMR with Structural and Biophysical Techniques to Design Challenging Protein‐Drug Conjugates

“…Furthermore, these systems are too big for NMR spectroscopy in solution, but still neither big nor rigid enough to allow for the use of cryo‐electron microscopy [27] . Solid‐state NMR may overcome these limitations, and it is already used to investigate non‐crystalline protein samples, biologics and biomaterials [28–39] . Significant enhancements in sensitivity have been obtained by the recent achievements in the NMR probe technology and in biomolecular Dynamic Nuclear Polarization (DNP) [40–44] …”

“…[27] Solid-state NMR may overcome these limitations, and it is already used to investigate noncrystalline protein samples, biologics and biomaterials. [28][29][30][31][32][33][34][35][36][37][38][39] Significant enhancements in sensitivity have been obtained by the recent achievements in the NMR probe technology and in biomolecular Dynamic Nuclear Polarization (DNP). [40][41][42][43][44] We here report the design and synthesis of a new molecule that results from the conjugation of the cytotoxic Paclitaxel with Tafamidis (Scheme 1) to form a stable noncovalent protein-drug conjugate (PDC) with TTR.…”

Multi-Functional Hydrogels Simultaneously Featuring Strong Fluorescence, Ultralong Phosphorescence, and Excellent Self-Healing Properties and Their Use for Advanced Anti-counterfeiting

“…Functional hydrogels have shown high promise for myriad applications, such as anti-counterfeiting, in wearable sensory devices and soft robots, − and so forth. Of particular note, fluorescent hydrogels have been recently employed for information encryption to take advantages of their good shape-memory ability and multiple responsiveness. − For instance, Wu et al introduced tough hydrogels with photo-tunable fluorescence, which enabled reprogrammable shape designing and information encoding .…”