Ellena, J. F. | bioRxiv Science

bioRxiv2024

Measurement of covalent bond formation in light-curing hydrogels predicts physical stability under flow

Photocrosslinking hydrogels are promising for tissue engineering and regenerative medicine, but challenges in reaction monitoring often leave their optimization subject to trial and error. The stability of crosslinked gels under fluid flow, as in the case of a microfluidic device, is particularly challenging to predict, both because of obstacles inherent to solid-state macromolecular analysis that prevent accurate chemical monitoring, and because stability is dependent on size of the patterned features. To solve both problems, we obtained 1H NMR spectra of cured hydrogels which were enzymatically degraded. This allowed us to take advantage of the high-resolution that solution NMR provides. This unique approach enabled the measurement of degree of crosslinking (DoC) and prediction of material stability under physiological fluid flow. We showed that NMR spectra of enzyme-digested gels successfully reported on DoC as a function of light exposure and wavelength within two classes of photocrosslinkable hydrogels: methacryloyl-modified gelatin and a composite of thiol-modified gelatin and norbornene-terminated polyethylene glycol. This approach revealed that a threshold DoC was required for patterned features in each material to become stable, and that smaller features required a higher DoC for stability. Finally, we demonstrated that DoC was predictive of the stability of architecturally complex features when photopatterning, underscoring the value of monitoring DoC when using light-reactive gels. We anticipate that the ability to quantify chemical crosslinks will accelerate the design of advanced hydrogel materials for structurally demanding applications such as photopatterning and bioprinting. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/601353v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1aa4a43org.highwire.dtl.DTLVardef@f1b008org.highwire.dtl.DTLVardef@15bf652org.highwire.dtl.DTLVardef@88a825_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Structural, in silico, and functional analysis of a Disabled-2-derived peptide for recognition of sulfatides

Disabled-2 (Dab2) is an adaptor protein that regulates numerous cellular processes. Among them, Dab2 modulates the extent of platelet aggregation by two mechanisms. In the first mechanism, Dab2 intracellularly downregulates the integrin IIb{beta}3 receptor, converting it to a low affinity state for adhesion and aggregation processes. In the second mechanism, Dab2 is released extracellularly and interacts with both the integrin IIb{beta}3 receptor and sulfatides, both of which are known to be pro-aggregatory mediators, blocking their association to fibrinogen and P-selectin, respectively. Our previous research indicated that a 35-amino acid region within Dab2, which we refer to as the sulfatide-binding peptide (SBP), contains two potential sulfatide-binding motifs represented by two consecutive polybasic regions. Using a combined methodology including molecular docking, nuclear magnetic resonance, lipid-binding assays, and surface plasmon resonance, this work identifies the critical Dab2 residues within SBP that are responsible for sulfatide binding. A hydrophilic region, primarily mediated by R42, is responsible for the interaction with the sulfatide headgroup, whereas the C-terminal polybasic region contributes to interactions with the acyl chains. Furthermore, we demonstrated that, in Dab2 SBP, R42 significantly contributes to the inhibition of platelet P-selectin surface expression. The interacting Dab2 SBP residues with sulfatide resemble those described for sphingolipid-binding in other proteins, suggesting that sulfatide-binding proteins share common binding mechanisms.

biophysics↗