title: Protein macromonomers containing reduction-sensitive linkers for covalent immobilization and glutathione triggered release from dextran hydrogels.
abstract: We report an efficient strategy to conjugate methacrylamide moieties to the lysine units of lysozyme for co-polymerization and subsequent triggered release from hydrogels. Two novel linker molecules, containing an ester bond and/or a disulfide bond for temporary immobilization, were synthesized and conjugated to lysozyme. Lysozyme was successfully modified with on average 2.5 linker molecules per protein molecule, as evidenced by MALDI-TOF and by titration of the free amine groups, while spectral analysis verified the preservation of the protein structure. Next, methacrylated dextran (Dex-MA) was polymerized in presence of native or modified lysozyme to yield hydrogels. The release of native and modified lysozyme from Dex-MA hydrogels was studied in acetate buffer (pH 5, in absence of any trigger) and only a minor fraction (~15%) of the modified lysozyme was released, whereas ~74% of the native lysozyme was released. This indicates successful immobilization of the majority of the modified lysozyme in the hydrogel network. Upon hydrolysis of the ester bonds or incubation with glutathione to reduce disulfide bonds of the linker molecules that conjugate the lysozyme to the gel network, the modified lysozyme was mobilized and released from the hydrogel to the same extent as native lysozyme. These data were confirmed by fluorescence recovery after photobleaching experiments. This approach appeared to be highly interesting for temporary immobilization and subsequent glutathione triggered intracellular delivery of proteins from hydrogels.
authors.affiliation: Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands.
authors.forename: Ellen
authors.initials: E
authors.lastname: Verheyen
authors.forename: Steffen
authors.initials: S
authors.lastname: van der Wal
authors.forename: Hendrik
authors.initials: H
authors.lastname: Deschout
authors.forename: Kevin
authors.initials: K
authors.lastname: Braeckmans
authors.forename: Stefaan
authors.initials: S
authors.lastname: de Smedt
authors.forename: Arjan
authors.initials: A
authors.lastname: Barendregt
authors.forename: Wim E
authors.initials: WE
authors.lastname: Hennink
authors.forename: Cornelus F
authors.initials: CF
authors.lastname: van Nostrum
mesh_terms: D000178:Acrylamides / Q000737:chemistry; D000818:Animals; D002645:Chickens; D003692:Delayed-Action Preparations / Q000737:chemistry*; D003911:Dextrans / Q000737:chemistry*; D004800:Enzymes, Immobilized / Q000008:administration & dosage* / Q000737:chemistry; D005978:Glutathione / Q000737:chemistry*; D020100:Hydrogels / Q000737:chemistry*; D006868:Hydrolysis; D008689:Methacrylates / Q000737:chemistry*; D009113:Muramidase / Q000008:administration & dosage* / Q000737:chemistry; D010084:Oxidation-Reduction; D058105:Polymerization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000178:Acrylamides; D003692:Delayed-Action Preparations; D003911:Dextrans; D004800:Enzymes, Immobilized; D020100:Hydrogels; D008689:Methacrylates; C514458:methacrylated dextran; D009113:Muramidase; D005978:Glutathione; C045985:methacrylamide