title: Sequential Thiol-Ene and Tetrazine Click Reactions for the Polymerization and Functionalization of Hydrogel Microparticles.
abstract: Click chemistry is a versatile tool for the synthesis and functionalization of polymeric biomaterials. Here, we describe a versatile new strategy for producing bioactive, protein-functionalized poly(ethylene glycol) (PEG) hydrogel microparticles that is based on sequential thiol-ene and tetrazine click reactions. Briefly, tetra-functional PEG-norbornene macromer and dithiothreitol (SH) cross-linker were combined at a 0.75:1 [SH]:[norbornene] ratio, emulsified in a continuous Dextran phase, and then photopolymerized to form PEG hydrogel microparticles that varied from 8 to 30 μm in diameter, depending on the PEG concentration used. Subsequently, tetrazine-functionalized protein was conjugated to unreacted norbornene groups in the PEG microparticles. Tetrazine-mediated protein tethering to the microparticles was first demonstrated using fluorescein-labeled ovalbumin as a model protein. Subsequently, bioactive protein tethering was demonstrated using alkaline phosphatase (ALP) and glucose oxidase (GOx). Enzyme activity assays demonstrated that both ALP and GOx maintained their bioactivity and imparted tunable bioactivity to the microparticles that depended on the amount of enzyme added. ALP-functionalized microparticles were also observed to initiate calcium phosphate mineralization in vitro when incubated with calcium glycerophosphate. Collectively, these results show that protein-functionalized hydrogel microparticles with tunable bioactive properties can be easily synthesized using sequential click chemistry reactions. This approach has potential for future applications in tissue engineering, drug delivery, and biosensing.
authors.affiliation: Department of Biomedical Engineering, ‡Department of Materials Science and Engineering, and §Center for Remote Health Technologies and Systems, Texas A&M University , College Station, Texas 77843, United States.
authors.forename: Faraz
authors.initials: F
authors.lastname: Jivan
authors.affiliation: Department of Biomedical Engineering, ‡Department of Materials Science and Engineering, and §Center for Remote Health Technologies and Systems, Texas A&M University , College Station, Texas 77843, United States.
authors.forename: Ramanathan
authors.initials: R
authors.lastname: Yegappan
authors.affiliation: Department of Biomedical Engineering, ‡Department of Materials Science and Engineering, and §Center for Remote Health Technologies and Systems, Texas A&M University , College Station, Texas 77843, United States.
authors.forename: Hannah
authors.initials: H
authors.lastname: Pearce
authors.affiliation: Department of Biomedical Engineering, ‡Department of Materials Science and Engineering, and §Center for Remote Health Technologies and Systems, Texas A&M University , College Station, Texas 77843, United States.
authors.forename: James K
authors.initials: JK
authors.lastname: Carrow
authors.affiliation: Department of Biomedical Engineering, ‡Department of Materials Science and Engineering, and §Center for Remote Health Technologies and Systems, Texas A&M University , College Station, Texas 77843, United States.
authors.forename: Michael
authors.initials: M
authors.lastname: McShane
authors.affiliation: Department of Biomedical Engineering, ‡Department of Materials Science and Engineering, and §Center for Remote Health Technologies and Systems, Texas A&M University , College Station, Texas 77843, United States.
authors.forename: Akhilesh K
authors.initials: AK
authors.lastname: Gaharwar
authors.affiliation: Department of Biomedical Engineering, ‡Department of Materials Science and Engineering, and §Center for Remote Health Technologies and Systems, Texas A&M University , College Station, Texas 77843, United States.
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Alge
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry*; D001672:Biocompatible Materials / Q000138:chemical synthesis / Q000737:chemistry / Q000627:therapeutic use; D057930:Click Chemistry; D016503:Drug Delivery Systems; D005949:Glucose Oxidase / Q000737:chemistry*; D006801:Humans; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000138:chemical synthesis / Q000737:chemistry* / Q000627:therapeutic use; D053758:Nanoparticles / Q000737:chemistry / Q000627:therapeutic use; D011092:Polyethylene Glycols / Q000138:chemical synthesis / Q000737:chemistry; D058105:Polymerization
chemical_list: D001672:Biocompatible Materials; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D011092:Polyethylene Glycols; D005949:Glucose Oxidase; D000469:Alkaline Phosphatase