Dataset Information

Bio-orthogonal conjugation and enzymatically triggered release of proteins within multi-layered hydrogels.

ABSTRACT: Hydrogels are facile architectures for the controlled presentation of proteins with far-reaching applications, from fundamental biological studies in three-dimensional culture to new regenerative medicine and therapeutic delivery strategies. Here, we demonstrate a versatile approach for spatially-defined presentation of engineered proteins within hydrogels through i) immobilization using bio-orthogonal strain-promoted alkyne-azide click chemistry and ii) dynamic protease-driven protein release using exogenously applied enzyme. Model fluorescent proteins were expressed using nonsense codon replacement to incorporate azide-containing unnatural amino acids in a site-specific manner toward maintaining protein activity: here, cyan fluorescent protein (AzCFP), mCherry fluorescent protein (AzmCh), and mCh decorated with a thrombin cut-site. (AzTMBmCh). Eight-arm poly(ethylene glycol) (PEG) was modified with dibenzylcyclooctyne (DBCO) groups and reacted with azide functionalized PEG in aqueous solution for rapid formation of hydrogels. Azide functionalized full-length fluorescent proteins were successfully incorporated into the hydrogel network by reaction with PEG-DBCO prior to gel formation. Temporal release and removal of select proteins (AzTMBmCh) was triggered with the application of thrombin and monitored in real-time with confocal microscopy, providing a responsive handle for controlling matrix properties. Hydrogels with regions of different protein compositions were created using a layering technique with thicknesses of hundreds of micrometers, affording opportunities for the creation of complex geometries on size scales relevant for controlling cellular microenvironments.

Statement of significance

Controlling protein presentation within biomaterials is important for modulating interactions with biological systems. For example, native tissues are composed of subunits with different matrix compositions (proteins, stiffness) that dynamically interact with cells, influencing function and fate. Toward mimicking such temporally-regulated and spatially-defined microenvironments, we utilize bio-orthogonal click chemistry and protein engineering to create hydrogels with distinct regions of proteins and modify them over time. Through nonsense codon replacement, we site-specifically functionalize large proteins with i) azides for covalent conjugation and ii) an enzymatic cleavage site for user-defined release from hydrogels. Our results exemplify not only the ability to create unique bio-functionalized hydrogels with controlled mechanical properties, but also the potential for creating interesting interfaces for cell culture and tissue engineering applications.

SUBMITTER: Guo C

PROVIDER: S-EPMC5510749 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Bio-orthogonal conjugation and enzymatically triggered release of proteins within multi-layered hydrogels.

Guo Chen C Kim Heejae H Ovadia Elisa M EM Mourafetis Christine M CM Yang Mingrui M Chen Wilfred W Kloxin April M AM

Acta biomaterialia 20170405

Hydrogels are facile architectures for the controlled presentation of proteins with far-reaching applications, from fundamental biological studies in three-dimensional culture to new regenerative medicine and therapeutic delivery strategies. Here, we demonstrate a versatile approach for spatially-defined presentation of engineered proteins within hydrogels through i) immobilization using bio-orthogonal strain-promoted alkyne-azide click chemistry and ii) dynamic protease-driven protein release u ...[more]

PMID: 28391052

Similar Datasets

Project description:In this work, we establish a quantitative correlation between molecular release and material degradation. We characterize a radical-initiated photopolymerized hydrogel and base-initiated Michael addition-polymerized hydrogel, which form gels through distinct crosslinking reactions. Both scaffolds use the same degradable peptide crosslinker, which enables them to be degraded through the same enzymatic degradation reaction. A fluorescently labeled poly(ethylene glycol) molecule is chemically conjugated into the scaffold and is released during enzymatic degradation. Real-time changes in scaffold rheological properties during degradation are measured using bulk rheology. Molecular release is measured by quantifying the change in fluorescence in the incubation liquid and the hydrogel scaffold. A complicating factor, previously described in the literature, is that shear may cause increased crosslinking, resulting in an increase in the storage modulus after initiation of degradation, which changes release profiles by limiting the initial release of molecules. Therefore, we also test the hypothesis that shear induces additional crosslinking in degrading hydrogel scaffolds. To determine whether shear changes rheological properties during scaffold degradation, enzymatic degradation is characterized using bulk rheology as materials undergo continuous or minimal shear. To determine the effect of shear on molecular release, shear is induced by shaking the material during incubation. Release is characterized from scaffolds that are incubated with continuous or without shaking. We determine that shear does not make a difference in scaffold degradation or release regardless of the gelation reaction. Instead, we determine that the type of hydrogel crosslinking reaction greatly affects both material degradation and molecular release. A hydrogel crosslinking by base-initiated Michael addition does undergo further crosslinking at the start of degradation. We correlate release with enzymatic degradation for both scaffolds. We determine that the material storage modulus is indirectly correlated with release during degradation. These results indicate that rheological characterization is a useful tool to characterize and predict the release of molecules from degrading hydrogels.

Dataset Information

Bio-orthogonal conjugation and enzymatically triggered release of proteins within multi-layered hydrogels.

Statement of significance

Publications

Bio-orthogonal conjugation and enzymatically triggered release of proteins within multi-layered hydrogels.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets