Structural analysis of photocrosslinkable methacryloyl-modified protein derivatives

Kan Yue; Xiuyu Li; Karsten Schrobback; Amir Sheikhi; Nasim Annabi; Jeroen Leijten; Weijia Zhang; Yu Shrike Zhang; Dietmar W Hutmacher; Travis J Klein; Ali Khademhosseini

doi:10.1016/j.biomaterials.2017.04.050

Structural analysis of photocrosslinkable methacryloyl-modified protein derivatives

Biomaterials. 2017 Sep:139:163-171. doi: 10.1016/j.biomaterials.2017.04.050. Epub 2017 May 29.

Authors

Affiliations

¹ Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, 02139, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
² Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, 02139, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA; Research Center for Analysis and Measurement, Hebei Normal University, Shijiazhuang, Hebei, 050024, China.
³ Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, 4059, Australia.
⁴ Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, 02139, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, 02115, USA.
⁵ Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, 02139, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, 02115, USA; Department of Chemical Engineering, Northeastern University, Boston, MA, 02115-5000, USA.
⁶ Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, 02139, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA; Department of Developmental BioEngineering, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands.
⁷ Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, 02139, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA; Department of Chemistry and Institute of Biomedical Science, Fudan University, Shanghai, 200433, China.
⁸ ARC Training Centre in Additive Biomanufacturing, Queensland University of Technology, Kelvin Grove, Queensland, 4059, Australia; Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, 4059, Australia.
⁹ Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, 4059, Australia; ARC Training Centre in Additive Biomanufacturing, Queensland University of Technology, Kelvin Grove, Queensland, 4059, Australia.
¹⁰ Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, 02139, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, 02115, USA; Department of Bioindustrial Technologies, College of Animal Bioscience and Technology, Konkuk University, Seoul, 143-701, Republic of Korea; Department of Physics, King Abdulaziz University, Jeddah, 21569, Saudi Arabia. Electronic address: [email protected].

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.

Keywords: GelMA; Gelatin; Protein modification; Structural analysis; Tropoelastin.

MeSH terms

Acrylamides / analysis
Acrylamides / chemistry*
Amines / chemistry
Biocompatible Materials / chemical synthesis
Biocompatible Materials / chemistry*
Chromatography, Liquid
Gelatin / chemistry*
Humans
Hydrogels / chemical synthesis
Hydrogels / chemistry
Hydroxides / chemistry
Hydroxylamine / chemistry
Iron / chemistry
Methacrylates / analysis
Methacrylates / chemistry*
Photochemical Processes
Proton Magnetic Resonance Spectroscopy
Tandem Mass Spectrometry
Tropoelastin / analysis
Tropoelastin / chemistry*

Substances

Acrylamides
Amines
Biocompatible Materials
Hydrogels
Hydroxides
Methacrylates
Tropoelastin
Hydroxylamine
Gelatin
hydroxide ion
Iron
methacrylamide

Abstract

MeSH terms

Substances

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