A Versatile Platform to Generate Shell-Cross-Linked Uniform Π-Conjugated Nanofibers with Controllable Length, High Morphological Stability, and Facile Surface Tailorability.

Living crystallization-driven self-assembly (CDSA) has emerged as an efficient route to generate π-conjugated-polymer-based nanofibers (CPNFs) with promising applications from photocatalysis to biomedicine. However, the lack of efficient tools to endow CPNFs with morphological stability and surface tailorability becomes a frustrating hindrance for expanding application spectrum of CPNFs. Herein, a facile strategy to fabricate length-controllable OPV-based (OPV = oligo(p-phenylenevinylene)) CPNFs containing a cross-linked shell with high morphological stability and facile surface tailorability through the combination of living CDSA and thiol-ene chemistry by using OPV5 -b-PNAAM32 (PNAAM = poly(N-allyl acrylamide)) as a model is reported. Uniform fiber-like micelles with tunable length can be generated by self-seeding of living CDSA. By taking advantage of radical thiol-ene reaction between vinyls of PNAAM corona and four-arm thiols, the shell of micelles can be cross-linked with negligible destruction of structure of vinylene-containing OPV core. The resulting micelles show high morphological stability in NaCl solution and PBS buffer, even upon heating at 80 °C. The introduced extra thiol groups in the cross-linked shell can be further employed to install extra functional moieties via convenient thiol-Michael-type reaction. Given the negligible cytotoxicity of resulting CPNFs, this strategy opens an avenue to fabricate various CPNFs of diverse functionalities for biomedicine.

Simple Preparation of Thiol-Ene Particles in Glycerol and Surface Functionalization by Thiol-Ene Chemistry (TEC) and Surface Chain Transfer Free Radical Polymerization (SCT-FRP).

Thiol-ene (TE)-based polymer particles are traditionally prepared via emulsion polymerization in water (using surfactants, stabilizers, and cosolvents). Here, a green and simple alternative is presented with excellent control over particle size, while avoiding the addition of stabilizers. Glycerol is applied as a dispersing medium for the preparation of off-stoichiometric TE microparticles, where sizes in the range of 40-400 µm are obtained solely by changing the mixing speed of the emulsions prior to crosslinking. Control over surface chemistry is achieved by surface functionalization of excess thiol groups via photochemical thiol-ene chemistry resulting in a functional monolayer. In addition, surface chain transfer free radical polymerization is used for the first time to introduce a thicker polymer layer on the particle surface. The application potential of the system is demonstrated by using functional particles as adsorbent for metal ions and as a support for immobilized enzymes.

Polythioether Particles Armored with Modifiable Graphene Oxide Nanosheets.

Facile and scalable fabrication methods are attractive to prepare materials for diverse applications. Herein, a method is presented to prepare cross-linked polymeric nanoparticles with graphene oxide (GO) nanosheets covalently attached to the surface. Alkene-modified GO serves as a surfactant in a miniemulsion polymerization, and the alkene functionalities of GO exposed to the oil-phase are incorporated into the polymer particle through thiol-ene reactions, leaving the unreacted alkene functional groups of the other face of GO available for further functionalization. The surface of GO-armored polymer particles is then modified with a small molecule fluorophore or carboxylic acid functional groups that bind to Fe2 O3 and TiO2 nanoparticles. This methodology provides a facile route to preparing complex hybrid composite materials.

Facile and Efficient Synthesis of Carbosiloxane Dendrimers via Orthogonal Click Chemistry Between Thiol and Ene.

A combination of a thiol-Michael addition reaction and a free radical mediated thiol-ene reaction is employed as a facile and efficient approach to carbosiloxane dendrimer synthesis. For the first time, carbosiloxane dendrimers are constructed rapidly by an orthogonal click strategy without protection/deprotection procedures. The chemoselectivity of these two thiol-ene click reactions leads to a design of a new monomer containing both electron-deficient carbon-carbon double bonds and unconjugated carbon-carbon double bonds. Siloxane bonds are introduced as the linker between these two kinds of carbon-carbon double bonds. Starting from a bifunctional thiol core, the dendrimers are constructed by iterative thiol-ene click reactions under different but both mild reaction conditions. After simple purification steps the fifth dendrimer with 54 peripheral functional groups is obtained with an excellent overall yield in a single day. Furthermore, a strong blue glow is observed when the dendrimer is excited by a UV lamp.

Ratiometric Fluorescent pH Probes Based on Glycopolymers.

Effectively detecting pH changes plays a critical role in exploring cellular functions and determining physiological and pathological processes. A novel ratiometric pH probe based on a glycopolymer, armored with properties of serum-stability, tumor-targeting, and pH monitoring, is designed. Random copolymers of 2-(methacrylamido) glucopyranose and fluorescein O-methacrylate are first synthesized by reversible addition fragmentation chain transfer polymerization. Acryloxyethyl thiocarbamoyl rhodamine B is then attached to the polymer chain to prepare ratiometric fluorescent pH probes via a thiol-ene reaction. The synthesized polymeric probes are characterized by NMR, gel permeation chromatography, UV-vis spectroscopy, and transmission electron microscopy, and the fluorescence responses are examined in phosphate buffer at different pHs. The cytotoxicity and confocal imaging experiments of the probes are detected using HeLa cells, demonstrating a low toxicity and superior biocompatibility for detecting pH changes in bioapplications.

Cysteine-functional polymers via thiol-ene conjugation.

A thiofunctional thiazolidine is introduced as a new low-molar-mass building block for the introduction of cysteine residues via a thiol-ene reaction. Allyl-functional polyglycidol (PG) is used as a model polymer to demonstrate polymer-analogue functionalization through reaction with the unsaturated side-chains. A modified trinitrobenzenesulfonic acid (TNBSA) assay is used for the redox-insensitive quantification and a precise final cysteine content can be predetermined at the polymerization stage. Native chemical ligation at cysteine-functional PG is performed as a model reaction for a chemoselective peptide modification of this polymer. The three-step synthesis of the thiofunctional thiazolidine reactant, together with the standard thiol-ene coupling and the robust quantification assay, broadens the toolbox for thiol-ene chemistry and offers a generic and straightforward approach to cysteine-functional materials.

Bioinspired polydopamine-mediated metal-organic framework click-grafting aptamers functionalized fabric for highly-specific recognition of microcystin-leucine arginine.

Fabricating functional electrospun nanofiber coating for highly selective extraction of microcystin-LR (MC-LR) was of significant importance for water-safety monitoring. Herein, a novel MOF@aptamer functionalized nanofabric was presented via a facile and reliable strategy integrating polydopamine (PDA) mediation and thiol-ene chemistry and applied for specific recognition of the MC-LR model analyte. Using polydopamine (PDA) as the mediating layer, vinyl-UiO-66 MOF was grown in situ, followed by post-synthetic modification (PSM) of Zr4+ with vinyl phosphate and rapid UV-initiated click reaction of aptamers. Uniform deposition of Zr-based MOF (vinyl-UiO-66) on the nanofibers was directly produced, and the tedious co-electrospinning process was abandoned to prevent the aggregation and encapsulation of MOF. Via an efficient "thiol-ene" chemistry, massive thiol-terminated aptamers were grafted on MOF within one step under friendly conditions, rather than the time-consuming nanoparticle adsorption or unfriendly covalent chemical reactions. As a result, the robust MOF@aptamer-coated nano-fabrics were obtained, and a highly selective performance towards MC-LR was illustrated with a limit of detection (LOD) at 0.002 ng/mL, good precision (CV<8.3%), good repeatability (2.2∼6.0%) when coupled with LC-MS. Almost 1∼2 orders of magnitude higher detection sensitivity was exhibited than that of the common non-specific SPE/SPME fiber reported so far. Applied to water samples, the good matrix-resistance ability, and acceptable recovery yields were achieved with high specificity. This strategy might provide a rapid and friendly protocol to efficiently fabricate MOF@aptamer functionalized nano-fabrics through electrospinning and interfacial "thiol-ene" chemistry for highly-selective microextraction.

Injectable thiol-ene hydrogel of galactoglucomannan and cellulose nanocrystals in delivery of therapeutic inorganic ions with embedded bioactive glass nanoparticles.

We propose an injectable nanocomposite hydrogel that is photo-curable via light-induced thiol-ene addition between methacrylate modified O-acetyl-galactoglucomannan (GGMMA) and thiolated cellulose nanocrystal (CNC-SH). Compared to free-radical chain polymerization, the orthogonal step-growth of thiol-ene addition allows a less heterogeneous hydrogel network and more rapid crosslinking kinetics. CNC-SH reinforced the GGMMA hydrogel as both a nanofiller and a crosslinker to GGMMA resulting in an interpenetrating network via thiol-ene addition. Importantly, the mechanical stiffness of the GGMMA/CNC-SH hydrogel is mainly determined by the stoichiometric ratio between the thiol groups on CNC-SH and the methacrylate groups in GGMMA. Meanwhile, the bioactive glass nanoparticle (BaGNP)-laden hydrogels of GGMMA/CNC-SH showed a sustained release of therapeutic ions in simulated body fluid in vitro, which extended the bioactive function of hydrogel matrix. Furthermore, the suitability of the GGMMA/CNC-SH formulation as biomaterial resin to fabricate digitally designed hydrogel constructs via digital light processing (DLP) lithography printing was evaluated.

Tuning Superfast Curing Thiol-Norbornene-Functionalized Gelatin Hydrogels for 3D Bioprinting.

Photocurable gelatin-based hydrogels have established themselves as powerful bioinks in tissue engineering due to their excellent biocompatibility, biodegradability, light responsiveness, thermosensitivity and bioprinting properties. While gelatin methacryloyl (GelMA) has been the gold standard for many years, thiol-ene hydrogel systems based on norbornene-functionalized gelatin (GelNB) and a thiolated crosslinker have recently gained increasing importance. In this paper, a highly reproducible water-based synthesis of GelNB is presented, avoiding the use of dimethyl sulfoxide (DMSO) as organic solvent and covering a broad range of degrees of functionalization (DoF: 20% to 97%). Mixing with thiolated gelatin (GelS) results in the superfast curing photoclick hydrogel GelNB/GelS. Its superior properties over GelMA, such as substantially reduced amounts of photoinitiator (0.03% (w/v)), superfast curing (1-2 s), higher network homogeneity, post-polymerization functionalization ability, minimal cross-reactivity with cellular components, and improved biocompatibility of hydrogel precursors and degradation products lead to increased survival of primary cells in 3D bioprinting. Post-printing viability analysis revealed excellent survival rates of > 84% for GelNB/GelS bioinks of varying crosslinking density, while cell survival for GelMA bioinks is strongly dependent on the DoF. Hence, the semisynthetic and easily accessible GelNB/GelS hydrogel is a highly promising bioink for future medical applications and other light-based biofabrication techniques.

Biomimetic stiffening of cell-laden hydrogels via sequential thiol-ene and hydrazone click reactions.

Hydrogels with dynamically tunable crosslinking are invaluable for directing stem cell fate and mimicking a stiffening matrix during fibrosis or tumor development. The increases in matrix stiffness during tissue development are often accompanied by the accumulation of extracellular matrices (e.g., collagen, hyaluronic acid (HA)), a phenomenon that has received little attention in the development of dynamic hydrogels. In this contribution, we present a gelatin-based cell-laden hydrogel system capable of being dynamically stiffened while accumulating HA, a key glycosaminoglycans (GAG) increasingly deposited by stromal cells during tumor progression. Central to this strategy is the synthesis of a dually-modified gelatin macromer - gelatin-norbornene-carbohydrazide (GelNB-CH), which is susceptible to both thiol-norbornene photopolymerization and hydrazone click chemistry. We demonstrate that the crosslinking density of cell-laden thiol-norbornene hydrogels can be dynamically tuned via simple incubation with aldehyde-bearing macromers (e.g., oxidized dextran (oDex) or oHA). The GelNB-CH hydrogel system is highly cytocompatible, as demonstrated by in situ encapsulation of pancreatic cancer cells (PCC) and cancer-associated fibroblasts (CAF). This unique dynamic stiffening scheme provides a platform to study tandem accumulation of HA and elevation in matrix stiffness in the pancreatic tumor microenvironment. STATEMENT OF SIGNIFICANCE: Hydrogels permitting on-demand and secondary crosslinking are ideal for mimicking a stiffening tumor microenvironment (TME). However, none of the current dynamic hydrogels account for both stiffening and accumulation of hyaluronic acid (HA), a major extracellular matrix component increasingly deposited in tumor stromal tissues, including pancreatic ductal adenocarcinoma (PDAC). The current work addresses this gap by developing a dynamic hydrogel system capable of simultaneously increasing stiffness and HA accumulation. This is achieved by a new gelatin macromer permitting sequential thiol-norbornene (for primary network crosslinking) and hydrazone click chemistry (for bioinert or biomimetic stiffening with oxidized dextran (oDex) or oHA, respectively). The results of this study provide new insights into how dynamically changing physicochemical matrix properties guide cancer cell fate processes.

Development of a Flow-free Gradient Generator Using a Self-Adhesive Thiol-acrylate Microfluidic Resin/Hydrogel (TAMR/H) Hybrid System.

Microfluidic gradient generators have been used to study cellular migration, growth, and drug response in numerous biological systems. One type of device combines a hydrogel and polydimethylsiloxane (PDMS) to generate "flow-free" gradients; however, their requirements for either negative flow or external clamps to maintain fluid-tight seals between the two layers have restricted their utility among broader applications. In this work, a two-layer, flow-free microfluidic gradient generator was developed using thiol-ene chemistry. Both rigid thiol-acrylate microfluidic resin (TAMR) and diffusive thiol-acrylate hydrogel (H) layers were synthesized from commercially available monomers at room temperature and pressure using a base-catalyzed Michael addition. The device consisted of three parallel microfluidic channels negatively imprinted in TAMR layered on top of the thiol-acrylate hydrogel to facilitate orthogonal diffusion of chemicals to the direction of flow. Upon contact, these two layers formed fluid-tight channels without any external pressure due to a strong adhesive interaction between the two layers. The diffusion of molecules through the TAMR/H system was confirmed both experimentally (using fluorescent microscopy) and computationally (using COMSOL). The performance of the TAMR/H system was compared to a conventional PDMS/agarose device with a similar geometry by studying the chemorepulsive response of a motile strain of GFP-expressing Escherichia coli. Population-based analysis confirmed a similar migratory response of both wild-type and mutant E. coli in both of the microfluidic devices. This confirmed that the TAMR/H hybrid system is a viable alternative to traditional PDMS-based microfluidic gradient generators and can be used for several different applications.

Thiol-norbornene gelatin hydrogels: influence of thiolated crosslinker on network properties and high definition 3D printing.

Photocrosslinkable gelatin hydrogels are excellent bioinks or biomaterial ink components to serve biofabrication applications. Especially the widely investigated gelatin-methacroyl (gel-MA) hydrogels hold an impressive track record. However, over the past decade, increasing attention is being paid to thiol-ene photo-click chemistry to obtain hydrogel networks benefitting from a faster reactivity (i.e. seconds vs minutes) along with superior biocompatibility and processability. In order to exploit this photo-click chemistry, often an ene-functionality (e.g. norbornene) is introduced onto gelatin followed by crosslinking in the presence of a multifunctional thiol (e.g. dithiothreitol). To date, very limited research has been performed on the influence of the applied thiolated crosslinker on the final hydrogel properties. Therefore, the present work assesses the influence of different thiolated crosslinkers on the crosslinking kinetics, mechanical properties and biological performance of the hydrogels upon encapsulation of primary adipose tissue-derived stem cells which indicated a cell viability exceeding 70%. Furthermore, the different formulations were processed using two-photon polymerization which indicated, in addition to differences in processing window and swelling ratio, a previously unreported phenomenon. At high intensities (i.e. ⩾150 mW), the laser results in cleavage of the gelatin backbone even in the absence of distinct photo-cleavable functionalities. This can have potential to introduce channels or softer regions in gels to result in zones characterized by different degradation speeds or the formation of blood vessels. Consequently, the present study can be used to provide guidance towards tailoring the thiol-ene system towards the desired applications.

Thiol-Gelatin-Norbornene Bioink for Laser-Based High-Definition Bioprinting.

Two-photon polymerization (2PP) is a lithography-based 3D printing method allowing the fabrication of 3D structures with sub-micrometer resolution. This work focuses on the characterization of gelatin-norbornene (Gel-NB) bioinks which enables the embedding of cells via 2PP. The high reactivity of the thiol-ene system allows 2PP processing of cell-containing materials at remarkably high scanning speeds (1000 mm s-1 ) placing this technology in the domain of bioprinting. Atomic force microscopy results demonstrate that the indentation moduli of the produced hydrogel constructs can be adjusted in the 0.2-0.7 kPa range by controlling the 2PP processing parameters. Using this approach gradient 3D constructs are produced and the morphology of the embedded cells is observed in the course of 3 weeks. Furthermore, it is possible to tune the enzymatic degradation of the crosslinked bioink by varying the applied laser power. The 3D printed Gel-NB hydrogel constructs show exceptional biocompatibility, supported cell adhesion, and migration. Furthermore, cells maintain their proliferation capacity demonstrated by Ki-67 immunostaining. Moreover, the results demonstrate that direct embedding of cells provides uniform distribution and high cell loading independently of the pore size of the scaffold. The investigated photosensitive bioink enables high-definition bioprinting of well-defined constructs for long-term cell culture studies.

Cross-Linked Polythiomethacrylate Esters Based on Naphthalene-Synthesis, Properties and Reprocessing.

Two structurally different aromatic dithioesters were synthesized from two dithiols and methacryloyl chloride. The polymer networks based on methyl methacrylate and/or styrene and the new dimethacrylates were subsequently prepared. The polymerization yields of copolymers were in the range of 95-99%. The thermal and mechanical properties of the copolymers were determined by means of differential scanning calorimetry (DSC), thermogravimetric analysis (TG/DTG), and Shore D hardness. The addition of dithioesters-1,5-NAF-S-Met (or 1,4(1,5)-NAF-CH2S-Met) (from 0.5% to 5%) to MMA- or ST-based polymers results in lowering the glass transition temperature (Tg) by about 8 °C. The thioester-containing polymers based on MMA exhibit lower thermal stability than those with ST. The polythioesters are stable up to 250 °C. The UV/vis spectra and refractive indexes of prepared liquid compositions were also measured. The 1,5-NAF-S-Met (and 1,4(1,5)-NAF-CH2S-Met) improved the refractive index values of ST and MMA compositions. The double bond conversion was also determined for all synthesized materials. The swelling studies of polymers with 20% addition of thioester crosslinkers were investigated. For all polymeric materials with 20% addition of thioesters, depolymerization of the network was carried out by thiol-thioester exchange. The depolymerization products were re-reacted in a thiol-ene reaction with 2-hydroxyethyl methacrylate by thermal initiation. The thiol-ene procedure enabled reprocessing of starting polymers and obtaining new materials characterized by distinctly different thermal, mechanical, and swelling properties. The thiol-ene materials exhibit a lower Shore hardness in the range of 20-50 °Sh, as well as decreased Tg values when compared to starting copolymers. Due to these possible exchange reactions, one can facilely manipulate the properties of the polymers which could lead to the manufacturing of the new products with the desired features. Degradation of the cross-linked structure and recycling of copolymers were also discussed.

Tuning Porosity and Functionality of Electrospun Rubber Nanofiber Mats by Photo-Crosslinking.

The present work proposes a versatile and efficient method to fabricate rubber nanofiber membranes with a controlled morphology and tailored functionality, based on the application of photoinduced thiol-ene cross-linking reactions to electrospun mats. Besides preventing the polymer cold flow and freezing the structure obtained by electrospinning, the photocuring step finely controls the morphology of the nanofiber mats, in terms of the fiber diameter up to the nanometer range and of the membrane porosity. Nanofiber membranes are also made chemically resistant, while retaining their flexibility. Finally, the proposed approach allows imparting specific functionalities to the rubber nanofibers: the type and concentration of the functional groups can be precisely tuned by changing process parameters (i.e., thiol/ene stoichiometric ratio and irradiation dose). Active chemical groups that remain available on the surface of the nanofibers can be used for further material modifications, as here proven by two target reactions. This key result is also demonstrated with electrospun membranes embedded into a microfluidic chip, opening the way to advanced functional flexible devices.

Enzymatically-degradable alginate hydrogels promote cell spreading and in vivo tissue infiltration.

Enzymatically-degradable materials recapitulate the dynamic and reciprocal interactions between cells and their native microenvironment by allowing cells to actively shape the degradation process. In order to engineer a synthetic 3D environment enabling cells to orchestrate the degradation of the surrounding material, norbornene-modified alginate was crosslinked with two different peptide crosslinkers susceptible to cleavage by matrix metalloproteinases using UV-initiated thiol-ene chemistry. Resulting hydrogels were characterized for their initial mechanical and rheological properties, and their degradation behavior was measured by tracking changes in wet weight upon enzyme incubation. This process was found to be a function of the crosslinker type and enzyme concentration, indicating that degradation kinetics could be controlled and tuned. When mouse embryonic fibroblasts were encapsulated in 3D, cell number remained constant and viability was high in all materials, while cell spreading and extensive filopodia formation was observed only in the degradable gels, not in non-degradable controls. After implanting hydrogels into the backs of C57/Bl6 mice for 8 weeks, histological stainings of recovered gel remnants and surrounding tissue revealed higher tissue and cell infiltration into degradable materials compared to non-degradable controls. This alginate-based material platform with cell-empowered enzymatic degradation could prove useful in diverse tissue engineering contexts, such as regeneration and drug delivery.

Development of a thiol-ene based screening platform for enzyme immobilization demonstrated using horseradish peroxidase.

Efficient immobilization of enzymes on support surfaces requires an exact match between the surface chemistry and the specific enzyme. A successful match would normally be identified through time consuming screening of conventional resins in multiple experiments testing individual immobilization strategies. In this study we present a versatile strategy that largely expands the number of possible surface functionalities for enzyme immobilization in a single, generic platform. The combination of many individual surface chemistries and thus immobilization methods in one modular system permits faster and more efficient screening, which we believe will result in a higher chance of discovery of optimal surface/enzyme interactions. The proposed system consists of a thiol-functional microplate prepared through fast photochemical curing of an off-stoichiometric thiol-ene (OSTE) mixture. Surface functionalization by thiol-ene chemistry (TEC) resulted in the formation of a functional monolayer in each well, whereas, polymer surface grafts were introduced through surface chain transfer free radical polymerization (SCT-FRP). Enzyme immobilization on the modified surfaces was evaluated by using a rhodamine labeled horseradish peroxidase (Rho-HRP) as a model enzyme, and the amount of immobilized enzyme was qualitatively assessed by fluorescence intensity (FI) measurements. Subsequently, Rho-HRP activity was measured directly on the surface. The broad range of utilized surface chemistries permits direct correlation of enzymatic activity to the surface functionality and improves the determination of promising enzyme-surface candidates. The results underline the high potential of this system as a screening platform for synergistic immobilization of enzymes onto thiol-ene polymer surfaces. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1267-1277, 2017.

Surface modification of model hydrogel contact lenses with hyaluronic acid via thiol-ene "click" chemistry for enhancing surface characteristics.

Discontinuation of contact lens wear as a result of ocular dryness and discomfort is extremely common; as many as 26% of contact lens wearers discontinue use within the first year. While patients are generally satisfied with conventional hydrogel lenses, improving on-eye comfort continues to remain a goal. Surface modification with a biomimetic, ocular friendly hydrophilic layer of a wetting agent is hypothesized to improve the interfacial interactions of the contact lens with the ocular surface. In this work, the synthesis and characterization of poly(2-hydroxyethyl methacrylate) surfaces grafted with a hydrophilic layer of hyaluronic acid are described. The immobilization reaction involved the covalent attachment of thiolated hyaluronic acid (20 kDa) on acrylated poly(2-hydroxyethyl methacrylate) via nucleophile-initiated Michael addition thiol-ene "click" chemistry. The surface chemistry of the modified surfaces was analyzed by Fourier transform infrared spectroscopy-attenuated total reflectance and X-ray photoelectron spectroscopy. The appearance of N (1s) and S (2p) peaks on the low resolution X-ray photoelectron spectroscopy spectra confirmed successful immobilization of hyaluronic acid. Grafting hyaluronic acid to the poly(2-hydroxyethyl methacrylate) surfaces decreased the contact angle, the dehydration rate, and the amount of nonspecific sorption of lysozyme and albumin in comparison to pristine hydrogel materials, suggesting the development of more wettable surfaces with improved water-retentive and antifouling properties, while maintaining optical transparency (>92%). In vitro testing also showed excellent viability of human corneal epithelial cells with the hyaluronic acid-grafted poly(2-hydroxyethyl methacrylate) surfaces. Hence, surface modification with hyaluronic acid via thiol-ene "click" chemistry could be useful in improving contact lens surface properties, potentially alleviating symptoms of contact lens related dryness and discomfort during wear.

Human iPSC-derived endothelial cell sprouting assay in synthetic hydrogel arrays.

UNLABELLED: Activation of vascular endothelial cells (ECs) by growth factors initiates a cascade of events during angiogenesis in vivo consisting of EC tip cell selection, sprout formation, EC stalk cell proliferation, and ultimately vascular stabilization by support cells. Although EC functional assays can recapitulate one or more aspects of angiogenesis in vitro, they are often limited by undefined substrates and lack of dependence on key angiogenic signaling axes. Here, we designed and characterized a chemically-defined model of endothelial sprouting behavior in vitro using human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs). We rapidly encapsulated iPSC-ECs at high density in poly(ethylene glycol) (PEG) hydrogel spheres using thiol-ene chemistry and subsequently encapsulated cell-dense hydrogel spheres in a cell-free hydrogel layer. The hydrogel sprouting array supported pro-angiogenic phenotype of iPSC-ECs and supported growth factor-dependent proliferation and sprouting behavior. iPSC-ECs in the sprouting model responded appropriately to several reference pharmacological angiogenesis inhibitors of vascular endothelial growth factor, NF-κB, matrix metalloproteinase-2/9, protein kinase activity, and ß-tubulin, which confirms their functional role in endothelial sprouting. A blinded screen of 38 putative vascular disrupting compounds from the US Environmental Protection Agency's ToxCast library identified six compounds that inhibited iPSC-EC sprouting and five compounds that were overtly cytotoxic to iPSC-ECs at a single concentration. The chemically-defined iPSC-EC sprouting model (iSM) is thus amenable to enhanced-throughput screening of small molecular libraries for effects on angiogenic sprouting and iPSC-EC toxicity assessment. STATEMENT OF SIGNIFICANCE: Angiogenesis assays that are commonly used for drug screening and toxicity assessment applications typically utilize natural substrates like Matrigel(TM) that are difficult to spatially pattern, costly, ill-defined, and may exhibit lot-to-lot variability. Herein, we describe a novel angiogenic sprouting assay using chemically-defined, bioinert poly(ethylene glycol) hydrogels functionalized with biomimetic peptides to promote cell attachment and degradation in a reproducible format that may mitigate the need for natural substrates. The quantitative assay of angiogenic sprouting here enables precise control over the initial conditions and can be formulated into arrays for screening. The sprouting assay here was dependent on key angiogenic signaling axes in a screen of angiogenesis inhibitors and a blinded screen of putative vascular disrupting compounds from the US-EPA.

Modular modification of xylan with UV-initiated thiol-ene reaction.

Birch xylan was functionalized with various thiols through UV initiated radical thiol-ene reaction under mild conditions. Xylan was allylated through etherification with allyl glycidyl ether under alkaline conditions. The allylated xylan was then reacted with thiols containing varying functional groups: trimethylbenzyl mercaptan, dodecanethiol, thioglycolic acid, L-cysteine and cysteamine hydrochloride. The reactions were conducted under homogeneous conditions at room temperature, either in water (hydrophilic thiols) or in DMF (hydrophobic thiols). The effect of reaction parameters to the functionalization efficiency was studied, including, for example, thiol excess, thiol character, initiator amount and reaction mixture concentration. The reactions were fast and 100% conversion of allyl groups was reached in most cases, sometimes already within 10 min. Water as solvent resulted generally in faster reactions when compared to DMF, and it was possible to conduct the aqueous reaction even without added UV initiator. It was also possible to incorporate two functionalities simultaneously during one reaction into the xylan structure.