Click chemistry reactions have become an important tool for synthesizing user-defined hydrogels consisting of poly(ethylene glycol) (PEG) and bioactive peptides for tissue engineering. However, because click crosslinking proceeds via a step-growth mechanism, multi-arm telechelic precursors are required, which has some disadvantages. Here, we report for the first time that this requirement can be circumvented to create PEG–peptide hydrogels solely from linear precursors through the use of two orthogonal click reactions, the thiol–maleimide Michael addition and thiol–norbornene click reaction. The rapid kinetics of both click reactions allowed for quick formation of norbornene-functionalized PEG–peptide block copolymers via Michael addition, which were subsequently photocrosslinked into hydrogels with a dithiol linker. Characterization and in vitro testing demonstrated that the hydrogels have highly tunable physicochemical properties and excellent cytocompatibility. In addition, stoichiometric control over the crosslinking reaction can be leveraged to leave unreacted norbornene groups in the hydrogel for subsequent hydrogel functionalization via bioorthogonal inverse-electron demand Diels–Alder click reactions with s -tetrazines. After selectively capping norbornene groups in a user-defined region with cysteine, this feature was leveraged for protein patterning. Collectively, these results demonstrate that our novel chemical strategy is a simple and versatile approach to the development of hydrogels for tissue engineering that could be useful for a variety of applications.
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High-throughput double emulsion-based microfluidic production of hydrogel microspheres with tunable chemical functionalities toward biomolecular conjugation
Chemically functional hydrogel microspheres hold significant potential in a range of applications including biosensing, drug delivery, and tissue engineering due to their high degree of flexibility in imparting a range of functions. In this work, we present a simple, efficient, and high-throughput capillary microfluidic approach for controlled fabrication of monodisperse and chemically functional hydrogel microspheres via formation of double emulsion drops with an ultra-thin oil shell as a sacrificial template. This method utilizes spontaneous dewetting of the oil phase upon polymerization and transfer into aqueous solution, resulting in poly(ethylene glycol) (PEG)-based microspheres containing primary amines (chitosan, CS) or carboxylates (acrylic acid, AA) for chemical functionality. Simple fluorescent labelling of the as-prepared microspheres shows the presence of abundant, uniformly distributed and readily tunable functional groups throughout the microspheres. Furthermore, we show the utility of chitosan's primary amine as an efficient conjugation handle at physiological pH due to its low pKa by direct comparison with other primary amines. We also report the utility of these microspheres in biomolecular conjugation using model fluorescent proteins, R-phycoerythrin (R-PE) and green fluorescent protein (GFPuv), via tetrazine– trans -cyclooctene (Tz–TCO) ligation for CS-PEG microspheres and carbodiimide chemistry for AA-PEG microspheres, respectively. The results show rapid coupling of R-PE with the microspheres' functional groups with minimal non-specific adsorption. In-depth protein conjugation kinetics studies with our microspheres highlight the differences in reaction and diffusion of R-PE with CS-PEG and AA-PEG microspheres. Finally, we demonstrate orthogonal one-pot protein conjugation of R-PE and GFPuv with CS-PEG and AA-PEG microspheres via simple size-based encoding. Combined, these results represent a significant advancement in the rapid and reliable fabrication of monodisperse and chemically functional hydrogel microspheres with tunable properties.
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- NSF-PAR ID:
- 10058427
- Date Published:
- Journal Name:
- Lab on a Chip
- Volume:
- 18
- Issue:
- 2
- ISSN:
- 1473-0197
- Page Range / eLocation ID:
- 323 to 334
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Cardiovascular disease is the leading cause of death worldwide, and current treatments are ineffective or unavailable to majority of patients. Engineered cardiac tissue (ECT) is a promising treatment to restore function to the damaged myocardium; however, for these treatments to become a reality, tissue fabrication must be amenable to scalable production and be used in suspension culture. Here, we have developed a low‐cost and scalable emulsion‐based method for producing ECT microspheres from poly(ethylene glycol) (PEG)–fibrinogen encapsulated mouse embryonic stem cells (mESCs). Cell‐laden microspheres were formed via water‐in‐oil emulsification; encapsulation occurred by suspending the cells in hydrogel precursor solution at cell densities from 5 to 60 million cells/ml, adding to mineral oil and vortexing. Microsphere diameters ranged from 30 to 570 μm; size variability was decreased by the addition of 2% poly(ethylene glycol) diacrylate. Initial cell encapsulation density impacted the ability for mESCs to grow and differentiate, with the greatest success occurring at higher cell densities. Microspheres differentiated into dense spheroidal ECTs with spontaneous contractions occurring as early as Day 10 of cardiac differentiation; furthermore, these ECT microspheres exhibited appropriate temporal changes in gene expression and response to pharmacological stimuli. These results demonstrate the ability to use an emulsion approach to encapsulate pluripotent stem cells for use in microsphere‐based cardiac differentiation.
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Mechanoresponsive, soft, photonic materials with tunable structural coloration represent a class of materials that have potential benefits for a wide range of applications. While many lab‐scale fabrication approaches afford control over the nano‐ and microscale morphology of these materials, upscaling their manufacture remains a challenge. Herein, a scalable fabrication concept is proposed that centers on the modular assembly of color‐changing materials from microscale building blocks. The building blocks consist of hydrogel‐based spherical photonic crystals. They are formed through a water‐in‐oil emulsification of nanoscale colloidal particles suspended in the aqueous phase. Once formed, the photonic crystal microspheres are then assembled into macroscale photonic materials, such as stretchable fibers or sheets. The resulting materials respond to a mechanical deformation with a reversible, dynamic change in color. Fabricated via a scalable, modular‐assembly approach, these mechanoresponsive photonic fibers and sheets, in turn, form a valuable building block for sensing systems or visual communication in healthcare, architecture, and consumer product design.
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Green chemistry-based non-isocyanate polyurethanes (NIPU) are synthesized and 3D-printed via rapid, projection photopolymerization into compliant mechanisms of 3D structure with spatially-localized material properties. Trimethylolpropane allyl ether-cyclic carbonate is used to couple the unique properties of two types of reaction chemistry: (1) primary diamine-cyclic carbonate ring-opening conjugation for supplanting conventional isocyanate-polyol reactions in creating urethane groups, with the additional advantage of enabling modular segment interchangeability within the diurethane prepolymers; and (2) thiol–ene (click) conjugation for non-telechelic, low monodispersity, quasi-crystalline-capable, and alternating step-growth co-photopolymerization. Fourier transform infrared spectroscopy is used to monitor the functional group transformation in reactions, and to confirm these process-associated molecular products. The extent of how these processes utilize molecular tunability to affect material properties were investigated through measurement-based comparison of the various polymer compositions: frequency-related dynamic mechanical analysis, tension-related elastic-deformation mechanical analysis, and material swelling analysis. Stained murine myoblasts cultured on NIPU slabs were evaluated via fluorescent microscopy for “green-chemistry” affects on cytocompatibility and cell adhesion to assess potential biofouling resistance. 3D multi-material structures with micro-features were printed, thus demonstrating the capability to spatially pattern different NIPU materials in a controlled manner and build compliant mechanisms.more » « less
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Self‐assembling filomicelles (FMs) are of great interest to nanomedicine due to their structural flexibility, extensive systemic circulation time, and amenability to unique “cylinder‐to‐sphere” morphological transitions. However, current fabrication techniques for preparing FMs are highly variable and difficult to scale. Herein, it is demonstrated that tetrablock copolymers composed of poly(ethylene glycol)‐
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/c7lc01088e
