Photo-acoustic Visible Protein Scaffolds via Diazonium Coupling

Protein-based biomaterials are invisible to most medical imaging modalities, owing to their physical similarity to native tissue. This hinders the non-invasive monitoring of correct placement, longitudinal retention, and material integrity or degradation over time. As an exemplar of this problem, hernia mesh implantation is the most widely performed surgical operation at >20 million/year globally, with a complication rate >10%, where poor mesh detectability affects clinical management.

As a potential solution, click chemistry offers a versatile route towards minimally modified proteins, facilitating both the incorporation of contrast agents for multi-modality imaging and functionalisation with bioactive molecules for enhanced therapy. However, most amino-acid-specific coupling reactions have been thus far limited to the modification of small soluble proteins, owing to challenges such as the aqueous stability of reactive groups, and limited diffusivity into macromolecular materials.

Here, we present, for the first time, a tyrosine-specific diazonium coupling approach for labelling pre-formed protein scaffolds, including a clinically approved hernia mesh based on decellularised dermis (Bard, Xenmatrix). Using 2-Methoxy-4-nitrobenzene-1-diazonium 5-sulfonaphthalene-1-sulfonate we demonstrate the efficient labelling of a collagen-based hernia mesh, thereby enhancing optical properties to facilitate image-guided surgery. Reaction conditions were optimised to give even labelling throughout the scaffold volume, which we confirmed on 10 µm sections and microscopy. The retention of material properties post labelling was confirmed using mechanical testing, alongside cell-growth assays for biocompatibility. Non-invasive in vivo detection after subcutaneous implantation in mice was shown using photo-acoustic imaging at 680nm, revealing the longitudinal placement of materials at high resolution up to 2 months post implantation. Histology, including H+E, was performed to confirm unaltered inflammatory response.

This is the first time diazonium coupling has been used for the uniform modification of whole pre-formed protein scaffolds, expanding its use beyond small soluble proteins. We used this to produce a photo-acoustic visible hernia mesh, enabling non-invasive in vivo imaging over a biologically relevant timeframe.