Poly(ethylene glycol)-based biofunctional hydrogels mediated by peroxidase-catalyzed cross-linking reactions (original) (raw)
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Horseradish peroxidase-catalyzed hydrogelation for biomedical applications
Biomaterials science, 2018
Hydrogels catalyzed by horseradish peroxidase (HRP) serve as an efficient and effective platform for biomedical applications due to their mild reaction conditions for cells, fast and adjustable gelation rate in physiological conditions, and an abundance of substrates as water-soluble biocompatible polymers. In this review, we highlight the tunable characteristics and use of the HRP-catalyzed hydrogels and provide a brief overview of various substrates employed in the HRP system for different biomedical applications of the resultant hydrogels. In addition, we discuss and summarize the biocompatibility, possible functionalization, and biofabrication process. Finally, the future prospective of the HRP crosslinking system is highlighted with biomedical applications.
Biomaterials Research, 2016
Background: Dityrosine crosslinking in proteins is a bioinspired method of forming hydrogels. This study compares oxidative enzyme initiators for their relative crosslinking efficiency and cytocompatibility using the same phenol group and the same material platform. Four common enzyme and enzyme-like oxidative initiators were probed for resulting material properties and cell viability post-encapsulation. Results: All four initiators can be used to form phenol-crosslinked hydrogels, however gelation rates are dependent on enzyme type, concentration, and the oxidant. Horseradish peroxidase (HRP) or hematin with hydrogen peroxide led to a more rapid poly (vinyl alcohol)-tyramine (PVA-Tyr) polymerization (10-60 min) because a high oxidant concentration was dissolved within the macromer solution at the onset of crosslinking, whereas laccase and tyrosinase require oxygen diffusion to crosslink phenol residues and therefore took longer to gel (2.5+ hours). The use of hydrogen peroxide as an oxidant reduced cell viability immediately post-encapsulation. Laccase-and tyrosinase-mediated encapsulation of cells resulted in higher cell viability immediately post-encapsulation and significantly higher cell proliferation after one week of culture. Conclusions: Overall this study demonstrates that HRP/H 2 O 2 , hematin/H 2 O 2 , laccase, and tyrosinase can create injectable, in situ phenol-crosslinked hydrogels, however oxidant type and concentration are critical parameters to assess when phenol crosslinking hydrogels for cell-based applications.
Biomacromolecules, 2013
As stem-cell-based therapies rapidly advance toward clinical applications, there is a need for cheap, easily manufactured, injectable gels that can be tailored to carry stem cells and impart function to such cells. Herein we describe a process for making hydrogels composed of hydroxyphenyl propionic acid (HPA) conjugated, branched poly(ethylene glycol) (PEG) via an enzyme mediated, oxidative cross-linking method. Functionalization of the branched PEG with HPA at varying degrees of substitution was confirmed via attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and 1 H NMR. The versatility of this hydrogel system was exemplified through variations in the degree of HPA substitution, polymer concentration, and the concentration of cross-linking reagents (horseradish peroxidase and H 2 O 2), which resulted in a range of mechanical properties and gelation kinetics for these gels. Cross-linking of the PEG−HPA conjugate with a recombinantly produced Fibronectin fragment (Type III domains 7−10) encouraged attachment and spreading of human mesenchymal stem cells (hMSCs) when assessed in both two-dimensional and three-dimensional formats. Interestingly, when encapsulated in both nonfunctionalized and functionalized cross-linked PEG−HPA gels, MSCs showed good viability over all time periods assessed. With tunable gelation kinetics and mechanical properties, these hydrogels provide a flexible in vitro cell culture platform that will likely have significant utility in tissue engineering as an injectable delivery platform for cells to sites of tissue damage.
Enzyme-Mediated Redox Initiation for Hydrogel Generation and Cellular Encapsulation
Biomacromolecules, 2009
A rapid, water-soluble enzyme-mediated radical chain initiation system involving glucose oxidase and Fe +2 generated hydrogels within minutes at 25°C and in ambient oxygen. The initiation components were evaluated for their effect on polymerization rates of hydroxyethyl acrylate-poly (ethylene glycol) 575 diacrylate comonomer solutions using near-infrared spectroscopy. Increasing glucose concentration increased polymerization rates until reaching a rate plateau above 1 × 10 −3 M of glucose. A square root dependence of the initial polymerization rate on Fe +2 concentration was observed between 1.0 × 10 −4 M and 5.0 × 10 −4 M of Fe +2 whereupon excess Fe +2 reduced final acrylate conversions. The glucose oxidase-mediated initiation system was employed for encapsulation of fibroblasts (NIH3T3s) into a poly(ethylene glycol) tetra-acrylate (M n~2 0,000) hydrogel scaffold demonstrating 96% (±3%) viability at 24 hours post-encapsulation. This first use of enzyme-mediated redox radical chain initiation for cellular encapsulation demonstrates polymerization of hydrogels in situ with kinetic control, minimal oxygen inhibition issues and utilization of low initiator concentrations.
Enzymatically cross-linked hyperbranched polyglycerol hydrogels as scaffolds for living cells
Biomacromolecules, 2014
Although several strategies are now available to enzymatically cross-link linear polymers to hydrogels for biomedical use, little progress has been reported on the use of dendritic polymers for the same purpose. Herein, we demonstrate that horseradish peroxidase (HRP) successfully catalyzes the oxidative cross-linking of a hyperbranched polyglycerol (hPG) functionalized with phenol groups to hydrogels. The tunable cross-linking results in adjustable hydrogel properties. Because the obtained materials are cytocompatible, they have great potential for encapsulating living cells for regenerative therapy. The gel formation can be triggered by glucose and controlled well under various environmental conditions.
Enzymatic Crosslinked Hydrogels for Biomedical Application
2021
Self-assembled structures mostly arises through enzyme-regulated phenomena in nature under persistent conditions. Enzymatic reactions are one of main biological processes in fabrication and construction of supramolecular hydrogel networks required for biomedical applications. The enzymatic processes provide a unique opportunity to integrate hydrogel formation. In most of cases, structure and substrates of hydrogels are adjusted by enzyme catalysis due to the chemo-, regio- and stereo-selectivity of enzymes. Hydrogels processed by using various enzyme schemes showed remarkable characteristics as dynamic frames for cells, bioactive molecules and drugs in biomedical applications. A novel class of enzyme-mediated crosslinking hydrogels mimics the extracellular matrices by displaying unique physicochemical properties and functionalities like water-retention capacity, drug loading ability, biodegradability, biocompatibility, biostability, bioactivity, optoelectronic properties, self-heali...
Bio-inspired synthetic hydrogels. Chemical and physical cross-linking approaches for 3D cell culture
Radboud University Nijmegen, 2020
List of abbreviations Chapter 1 Biomimetic functional hydrogels for cell culture: from 2D to 3D Chapter 2 Peptide-functionalized PEG hydrogel for 2D and 3D cell culture Chapter 3 Comparison of bioorthogonally cross-linked hydrogels for in situ cell encapsulation Chapter 4 A hybrid peptide amphiphile fiber PEG hydrogel matrix for 3D cell culture Chapter 5 Self-healing dual cross-linked hydrogels based on bioorthogonal click chemistry and ionic interactions
Enzymatic Cross-Linking of Dynamic Thiol-Norbornene Click Hydrogels
ACS Biomaterials Science & Engineering, 2019
Enzyme-mediated in situ forming hydrogels are attractive for many biomedical applications because gelation afforded by the enzymatic reactions can be readily controlled not only by tuning macromer compositions, but also by adjusting enzyme kinetics. For example, horseradish peroxidase (HRP) has been used extensively for in situ crosslinking of macromers containing hydroxyl-phenol groups. The use of HRP on initiating thiol-allylether polymerization has also been reported, yet no prior study has demonstrated enzymatic initiation of thiol-norbornene gelation. In this study, we discovered that HRP can generate thiyl radicals needed for initiating thiol-norbornene hydrogelation, which has only been demonstrated previously using photopolymerization. Enzymatic thiol-norbornene gelation not only overcomes light attenuation issue commonly observed in photopolymerized hydrogels, but also preserves modularity of the crosslinking. In particular, we prepared modular hydrogels from two sets of norbornene-modified macromers, 8-arm poly(ethylene glycol)-norbornene (PEG8NB) and gelatin-norbornene (GelNB). Bis-cysteine-containing peptides or PEG-tetra-thiol (PEG4SH) were used as crosslinkers for forming enzymatically and orthogonally polymerized hydrogels. For HRP-initiated PEG-peptide hydrogel crosslinking, gelation efficiency was significantly improved via adding tyrosine residues on the peptide crosslinkers. Interestingly, these additional tyrosine residues did not form permanent dityrosine crosslinks following HRP-induced gelation. As a result, they remained available for tyrosinase-mediated secondary crosslinking, which dynamically increases hydrogel stiffness. In addition to material characterizations, we also found that both PEG- and gelatin-based hydrogels provide excellent cytocompatibility for dynamic 3D cell culture. The enzymatic thiol-norbornene gelation scheme presented here offers a new crosslinking mechanism for preparing modularly and dynamically crosslinked hydrogels.
Formation of Three-Dimensional Hydrogel Multilayers Using Enzyme-Mediated Redox Chain Initiation
ACS Applied Materials & Interfaces, 2010
Enzyme-mediated redox chain initiation involving glucose oxidase (GOX) was employed in an iterative solution dip-coating technique to polymerize multiple, three-dimensional hydrogel layers using mild aqueous conditions at ambient temperature and oxygen levels. To our knowledge, sequential enzyme-mediated dip-coating resulting in an interfacial radical chain polymerization and subsequent formation of three-dimensional hydrogel layers has not been previously explored. Conformal, micron-scale, uniform poly(ethylene glycol) (PEG)-based hydrogel layers were polymerized within seconds and remained securely associated after incubation in water for 16 weeks. Incorporation of either small molecules (i.e., rhodamine-B acrylate, fluorescein acrylate) or fluorescent nanoparticles into crosslinked hydrogel layers during the polymerization reaction was also achieved. The encapsulation of 0.2 μmdiameter nanoparticles into hydrogels during polymerization of a 2-hydroxyethyl acrylate (HEA)/PEG 575 diacrylate monomer formulation, using the GOX-mediated initiation, resulted in minimal effects on polymerization kinetics, with final acrylate conversions of 95% (±1%) achieved within minutes. The temporal control and spatial localization afforded by this interfacial redox approach resulted in the polymerization of uniform secondary layers ranging between 150 (±10) μm and 650 (±10) μm for 15 and 120 s immersion times, respectively. Moreover, increasing the PEG 575-fraction within the initial hydrogel substrate from 10% to 50% decreased the subsequent layer thicknesses from 690 (±30) μm to 490 (±10) μm owing to lowered glucose concentration at the hydrogel interface. The ability to sequentially combine differing initiation mechanisms with this coating approach was achieved by using GOX-mediated interfacial polymerization on hydrogel substrates initially photopolymerized in the presence of glucose. The strict control of layer thicknesses combined with the rapid, water soluble, and mild polymerization will readily benefit applications requiring formation of stratified, complex, and threedimensional polymer structures.