Thiol-ene radical coupling is increasingly used for the biofunctionalization of biomaterials and the formation of 3D hydrogels enabling cell encapsulation. Indeed, thiol-ene chemistry presents interesting features that are particularly attractive for platforms requiring specific reactions of peptides or proteins, in particular, in situ, during cell culture or encapsulation. Despite such interest, little is known about the factors impacting thiol-ene chemistry in situ, under biologically relevant conditions. Here we explore some of the molecular parameters controlling photoinitiated thiol-ene couplings with a series of alkenes and thiols, including peptides, in buffered conditions. (1)H NMR and HPLC were used to quantify the efficiency of couplings and the impact of the pH of the buffer, as well as the molecular structure and local microenvironment close to alkenes and thiols to be coupled. Some of these observations are supported by molecular dynamics and quantum mechanics calculations. An important finding of our work is that the pKa of thiols (and its variation upon changes in molecular structure) have a striking impact on coupling efficiencies. Similarly, positively charged and aromatic amino acids are found to have some impact on thiol-ene couplings. Hence, our study demonstrates that molecular design should be carefully selected in order to achieve high biofunctionalization levels in biomaterials with peptides or promote the efficient formation of peptide-based hydrogels.
The functionalisation and patterning of polymer brushes via thiol–ene chemistry is studied via ellipsometry, XPS and AFM.
Thiol-ene radical coupling is increasingly used for the biofunctionalisation of biomaterials and the formation of 3D hydrogels enabling cell encapsulation. Indeed, thiol-ene chemistry presents interesting features that are particularly attractive for platforms requiring specific reactions of peptides or proteins, in particular in situ, during cell culture or encapsulation: thiol-ene coupling occurs specifically between a thiol and a non-activated alkene (unlike Michael addition); it is relatively tolerant to the presence of oxygen; it can be triggered by light. Despite such interest, little is known about the factors impacting polymer thiol-ene chemistry in situ. Here we explore some of the molecular parameters controlling photoinitiated thiol-ene coupling (with UV and visible light irradiation), with a series of alkenefunctionalised polymer backbones. 1 H NMR spectroscopy is used to quantify the efficiency of couplings, whereas photo-rheology allows correlation to gelation and mechanical properties of the resulting materials. We identify the impact of weak electrolytes in regulating coupling efficiency, presumably via thiol deprotonation and regulation of local diffusion. The conformation of associated polymer chains, regulated by the pH, is also proposed to play an important role in the modulation of both thiol-ene coupling and crosslinking efficiencies. Ultimately, suitable conditions for cell encapsulations are identified for a range of polymer backbones and their impact on cytocompatibility is investigated for cell encapsulation and tissue engineering applications. Overall our work demonstrates the importance of polymer backbone design to regulate thiol-ene coupling and in situ hydrogel formation.
Thiol-ene radical coupling is increasingly used for the biofunctionalization of biomaterials. Thiol-ene chemistry presents interesting features that are particularly attractive for platforms requiring specific reactions with peptides or proteins and the patterning of cells, such as reactivity in physiological conditions and photoactivation. In this work, we synthesized alkene-functionalized (allyl and norbornene residues) antifouling polymer brushes (based on poly(oligoethylene glycol methacrylate)) and studied thiol-ene coupling with a series of thiols including cell adhesive peptides RGD and REDV. The adhesion of umbilical vein endothelial cells (HUVECs) to these interfaces was studied and highlighted the absence of specific integrin engagement to REDV, in contrast to the high level of cell spreading observed on RGD-functionalized polymer brushes. This revealed that αβ integrins (binding to REDV sequences) are not sufficient on their own to sustain HUVEC spreading, in contrast to αβ and αβ integrins. In addition, we photopatterned peptides at the surface of poly(oligoethylene glycol methacrylate) (POEGMA) brushes and characterized the quality of the resulting arrays by epifluorescence microscopy and atomic force microscopy (AFM). This allowed the formation of cell patterns and demonstrated the potential of thiol-ene based photopatterning for the design of cell microarrays.
Peptide cross-linked poly(ethylene glycol) hydrogel has been widely used for drug delivery and tissue engineering. However, the use of this material as a biosensor for the detection of collagenase has not been explored. Proteases play a key role in the pathology of diseases such as rheumatoid arthritis and osteoarthritis. The detection of this class of enzyme using the degradable hydrogel film format is promising as a point-of-care device for disease monitoring. In this study, a protease biosensor was developed based on the degradation of a peptide cross-linked poly(ethylene glycol) hydrogel film and demonstrated for the detection of collagenase. The hydrogel was deposited on gold-coated quartz crystals, and their degradation in the presence of collagenase was monitored using a quartz crystal microbalance (QCM). The biosensor was shown to respond to concentrations between 2 and 2000 nM in less than 10 min with a lower detection limit of 2 nM.
Inflammatory conditions are frequently accompanied by increased levels of active proteases, and there is rising interest in methods for their detection to monitor inflammation in a point of care setting. In this work, new sensor materials for disposable single-step protease biosensors based on poly(2-oxazoline) hydrogels cross-linked with a protease-specific cleavable peptide are described. The performance of the sensor material was assessed targeting the detection of matrix metalloproteinase-9 (MMP-9), a protease that has been shown to be an indicator of inflammation in multiple sclerosis and other inflammatory conditions. Films of the hydrogel were formed on gold-coated quartz crystals using thiol−ene click chemistry, and the cross-link density was optimized. The degradation rate of the hydrogel was monitored using a quartz crystal microbalance (QCM) and showed a strong dependence on the MMP-9 concentration. A concentration range of 0−160 nM of MMP-9 was investigated, and a lower limit of detection of 10 nM MMP-9 was determined.
Photocured silicone elastomers have become increasingly popular in a variety of advanced materials applications including soft robotics, biomedical devices, microfabrication, functional coatings, and additive manufacturing. Oxygen inhibition, however, remains a major challenge for conventional photoradical curing, limiting applications and restricting processing. Here, we report a thiol−norbornene-based silicone system using a polydimethylsiloxane (PDMS) displaying terminal norbornene groups and a highly functionalized thiolated PDMS to achieve very fast curing speeds in an ambient atmosphere. Complete curing, without any unreacted oily residue on the surface of the silicone elastomer, was achieved in the absence of inert atmosphere protection. The impact of a formulation and photoinitiating system, including the use of visible-light initiators, on the kinetics and mechanical properties of the silicone networks was studied. Despite their opacity, nanocomposites incorporating fumed silica and graphene oxide (GO) retained fast cure rates (gelation times below 1 and 5 s for silica and GO composites, respectively) with tack-free surfaces. After thermal conversion, this afforded composites with conductivities above 0.1 S/m. Finally, combination with a conventional room-temperature vulcanization system enabled the formulation of effective dual-cure composites. The fast crosslinking thiol−norbornene silicones reported are attractive for a wide range of applications where ambient curing is required or where processing requires ultrafast reaction rates.
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