Browsing by Author "Nguyen, Han D."

Now showing 1 - 6 of 6
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    Dynamic PEG-Peptide Hydrogels via Visible Light and FMN-Induced Tyrosine Dimerization
    (Wiley, 2018) Liu, Hung-Yi; Nguyen, Han D.; Lin, Chien-Chi; Biomedical Engineering, School of Engineering and Technology
    Photo-responsive hydrogels have become invaluable three-dimensional (3D) culture matrices for mimicking aspects of extracellular matrix (ECM). Recent efforts have focused on using ultraviolet (UV) light exposure and multifunctional macromers to induce secondary hydrogel crosslinking and dynamic matrix stiffening in the presence of cells. This contribution reports the design of a novel yet simple dynamic poly(ethylene glycol)-peptide hydrogel system through flavin mononucleotide (FMN) induced di-tyrosine crosslinking. These di-tyrosine linkages effectively increase hydrogel crosslinking density and elastic modulus. In addition, the degree of stiffening in hydrogels at a fixed PEG macromer content can be readily tuned by controlling FMN concentration or the number of tyrosine residues built-in to the peptide linker. Furthermore, tyrosine-bearing pendant biochemical motifs could be spatial-temporally patterned in the hydrogel network via controlling light exposure through a photomask. The visible light and FMN induced tyrosine dimerization process produces cytocompatible and physiologically relevant degree of stiffening, as shown by changes of cell morphology and gene expression in pancreatic cancer and stromal cells. This new dynamic hydrogel scheme should be highly desirable for researchers seeking a photo-responsive hydrogel system without complicated chemical synthesis and secondary UV light irradiation.
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    Enzymatic Cross-Linking of Dynamic Thiol-Norbornene Click Hydrogels
    (ACS, 2019-03-11) Nguyen, Han D.; Liu, Hung-Yi; Hudson, Britney N.; Lin, Chien-Chi; Biomedical Engineering, School of Engineering and Technology
    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.
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    Enzymatic Cross-Linking of Dynamic Thiol-Norbornene Click Hydrogels
    (ACS, 2019) Nguyen, Han D.; Liu, Hung-Yi; Hudson, Britney N.; Lin, Chien-Chi; Biomedical Engineering, School of Engineering and Technology
    Enzyme-mediated in situ forming hydrogels are attractive for many biomedical applications because gelation afforded by 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 cross-linking of macromers containing hydroxyl-phenol groups. The use of HRP to initiate 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 the 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 cross-linking. 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) was used as a cross-linker for forming enzymatically and orthogonally polymerized hydrogel. For HRP-initiated PEG-peptide hydrogel cross-linking, gelation efficiency was significantly improved via adding tyrosine residues on the peptide cross-linkers. Interestingly, these additional tyrosine residues did not form permanent dityrosine cross-links following HRP-induced gelation. As a result, they remained available for tyrosinase-mediated secondary cross-linking, which dynamically increased hydrogel stiffness. In addition to material characterizations, we also found that both PEG- and gelatin-based hydrogels exhibited excellent cytocompatibility for dynamic 3D cell culture. The enzymatic thiol-norbornene gelation scheme presented here offers a new cross-linking mechanism for preparing modularly and dynamically cross-linked hydrogels.
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    Probing osteocyte function in gelatin hydrogels with tunable viscoelasticity
    (American Chemical Society, 2021) Nguyen, Han D.; Sun, Xun; Yokota, Hiroki; Lin, Chien-Chi; Biomedical Engineering, School of Engineering and Technology
    Bone is an attractive site for metastatic cancer cells and has been considered as "soil" for promoting tumor growth. However, accumulating evidence suggests that some bone cells (e.g., osteocytes) can actually suppress cancer cell migration and invasion via direct cell-cell contact and/or through cytokine secretion. Toward designing a biomimetic niche for supporting 3D osteocyte culture, we present here a gelatin-based hydrogel system with independently tunable matrix stiffness and viscoelasticity. In particular, we synthesized a bifunctional macromer, gelatin-norbornene-boronic acid (i.e., GelNB-BA), for covalent cross-linking with multifunctional thiol linkers [e.g., four-arm poly(ethylene glycol)-thiol or PEG4SH] to form thiol-NB hydrogels. The immobilized BA moieties in the hydrogel readily formed reversible boronate ester bonds with 1,3-diols on physically entrapped poly(vinyl alcohol) (PVA). Adjusting the compositions of GelNB-BA, PEG4SH, and PVA afforded hydrogels with independently tunable elasticity and viscoelasticity. With this new dynamic hydrogel platform, we investigated matrix mechanics-induced growth and cytokine secretion of encapsulated MLO-A5 pre-osteocytes. We discovered that more compliant or viscoelastic gels promoted A5 cell growth. On the other hand, cells encapsulated in stiffer gels secreted higher amounts of pro-inflammatory cytokines and chemokines. Finally, conditioned media (CM) collected from the encapsulated MLO-A5 cells (i.e., A5-CM) strongly inhibited breast cancer cell proliferation, invasion, and expression of tumor-activating genes. This new biomimetic hydrogel platform not only serves as a versatile matrix for investigating mechano-sensing in osteocytes but also provides a means to produce powerful anti-tumor CM.
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    Recent advances in bio-orthogonal and dynamic crosslinking of biomimetic hydrogels
    (Royal Society of Chemistry, 2020-09-21) Arkenberg, Matthew R.; Nguyen, Han D.; Lin, Chien-Chi; Biomedical Engineering, School of Engineering and Technology
    In recent years, dynamic, 'click' hydrogels have been applied in numerous biomedical applications. Owing to the mild, cytocompatible, and highly specific reaction kinetics, a multitude of orthogonal handles have been developed for fabricating dynamic hydrogels to facilitate '4D' cell culture. The high degree of tunability in crosslinking reactions of orthogonal 'click' chemistry has enabled a bottom-up approach to install specific biomimicry in an artificial extracellular matrix. In addition to click chemistry, highly specific enzymatic reactions are also increasingly used for network crosslinking and for spatiotemporal control of hydrogel properties. On the other hand, covalent adaptable chemistry has been used to recapitulate the viscoelastic component of biological tissues and for formulating self-healing and shear-thinning hydrogels. The common feature of these three classes of chemistry (i.e., orthogonal click chemistry, enzymatic reactions, and covalent adaptable chemistry) is that they can be carried out under ambient and aqueous conditions, a prerequisite for maintaining cell viability for in situ cell encapsulation and post-gelation modification of network properties. Due to their orthogonality, different chemistries can also be applied sequentially to provide additional biochemical and mechanical control to guide cell behavior. Herein, we review recent advances in the use of orthogonal click chemistry, enzymatic reactions, and covalent adaptable chemistry for the development of dynamically tunable and biomimetic hydrogels.
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    Thiol-Norbornene Hydrogels With Tunable Mechanical Properties for Engineered Extracellular Matrices
    (2019-05) Nguyen, Han D.; Lin, Chien-Chi; Xie, Dong; Yokota, Hiroki
    The extracellular matrix (ECM) governs many cellular processes through biochemical and mechanical cues. Particularly, the effect ECM mechanical properties on cells fate has been well established over the years. Many hydrogel systems have been used to mimic the dynamic stiffening processes occurring in ECM. However, changes in ECM stiffness does not fully recapitulate the mechanics of native ECM, as viscoelasticity is also a major factor contributing to ECM dynamic property. This thesis describes the design and characterization of an enzyme-crosslinked hydrogel system that is not only capable of being stiffened on demand, but also can be tuned to obtain viscoelasticity. The first objective of this thesis was to utilize horseradish peroxidase (HRP) to crosslink thiol-norbornene hydrogel and use mushroom tyrosinase (MT) to create secondary DOPA-dimer crosslinks that stiffened the hydrogel. The cytocompatibility of HRP-mediated thiol-norbornene gelation and the effect of stiffening on cell fate was evaluated. The second objective of this thesis represented the first step towards developing a hydrogel system whose viscoelasticity could be dynamically tuned. Thiol-norbornene hydrogel was designed to yield dynamically adaptable boronic ester bonds via partial enzymatic reaction. Thiol-norborne hydrogel was made to contain hydroxyl phenol as well as boronic acid residues within its network. MT, in this case was used to oxidize the hydroxy phenol moieties into DOPA, which then complexed with boronic acid, created dynamic bonds, introducing viscoelasticity to an initial elastic hydrogel.