Structural analysis of photocrosslinkable methacryloyl-modified protein derivatives

Abstract

Biochemically modified proteins have attracted significant attention due to their widespread applications as biomaterials. For instance, chemically modified gelatin derivatives have been widely explored to develop hydrogels for tissue engineering and regenerative medicine applications. Among the reported methods, modification of gelatin with methacrylic anhydride (MA) stands out as a convenient and efficient strategy to introduce functional groups and form hydrogels via photopolymerization. Combining light-activation of modified gelatin with soft lithography has enabled the materialization of microfabricated hydrogels. So far, this gelatin derivative has been referred to in the literature as gelatin methacrylate, gelatin methacrylamide, or gelatin methacryloyl, with the same abbreviation of GelMA. Considering the complex composition of gelatin and the presence of different functional groups on the amino acid residues, both hydroxyl groups and amine groups can possibly react with methacrylic anhydride during functionalization of the protein. This can also apply to the modification of other proteins, such as recombinant human tropoelastin to form MA-modified tropoelastin (MeTro). Here, we employed analytical methods to quantitatively determine the amounts of methacrylate and methacrylamide groups in MA-modified gelatin and tropoelastin to better understand the reaction mechanism. By combining two chemical assays with instrumental techniques, such as proton nuclear magnetic resonance (1H NMR) and liquid chromatography tandem-mass spectrometry (LC-MS/MS), our results indicated that while amine groups had higher reactivity than hydroxyl groups and resulted in a majority of methacrylamide groups, modification of proteins by MA could lead to the formation of both methacrylamide and methacrylate groups. It is therefore suggested that the standard terms for GelMA and MeTro should be defined as gelatin methacryloyl and methacryloyl-substituted tropoelastin, respectively, to remain consistent with the widespread abbreviations used in literature.

Beyond the Modification Degree: Impact of Raw Material on Physicochemical Properties of Gelatin Type A and Type B Methacryloyls

“… The methacrylamide amount remained almost constant in all gelatin derivatives, while the amount of methacrylate groups rose with increasing excess of MAAnh. This observation was in good agreement to the absence of the lysine signal in all gelatin derivatives spectra and particularly reflects the elevated reactivity of amino functions compared to hydroxyl functions, as described before . The trend of declining DMA with increasing ratio of AcAnh for all derivatives that were reacted with a tenfold excess of anhydride (GM10, GM5A5, GM2A8) can be attributed to elevated hydrolysis sensitivity of AcAnh compared to MAAnh and thus less available anhydride in the course of the modification reaction when AcAnh was applied instead of MAAnh.…”

“…We characterized the G A and G B raw material by amino acid analysis (methods and results are given in Table S1, Supporting Information). It was shown recently by 2D‐NMR and liquid chromatography tandem‐mass spectrometry (LC‐MS/MS) that besides amino acids bearing amino groups also hydroxyl group containing amino acids were chemically modified during GM(A) synthesis. The amount of amino group containing amino acids (lysine and hydroxylysine, G A : 0.33 mmol g −1 , G B : 0.31 mmol g −1 ) and of hydroxyl group containing amino acids (serine, threonine, tyrosine, hydroxyproline, and hydroxylysine, G A : 1.47 mmol g −1 , G B : 1.50 mmol g −1 ) was almost the same for the used G A and G B .…”

Development of a Photo-Crosslinking, Biodegradable GelMA/PEGDA Hydrogel for Guided Bone Regeneration Materials

“…Since then, GelMA has been widely studied as a biomaterial with attractive properties [19,20]. GelMA hydrogel is prepared by the photocrosslinking method, which has the advantages of an injectable, mild crosslinking condition and low cytotoxicity [1].…”

Biomaterial‐based possibilities for managing peri‐implantitis

“…These biomaterials form a strong cross‐linked network of natural or synthetic molecules capable of storing biological drugs on their internal spaces. Among biomaterials used for hydrogel fabrication, polysaccharides (eg, dextran and chitosan) and proteins (eg, gelatin and fibrin) are well‐studied standards of natural polymers . Regarding synthetic biomaterials, polyvinyl alcohol (PVA), polyethylene glycol (PEG), and poly(acrylic acid) (PAA) are widely used examples of hydrogel‐forming polymers.…”