Hydrogel Cross-Linking via Thiol-Reactive Pyridazinediones

Thiol-reactive Michael acceptors are commonly used for the formation of chemically cross-linked hydrogels. In this paper, we address the drawbacks of many Michael acceptors by introducing pyridazinediones as new cross-linking agents. Through the use of pyridazinediones and their mono- or dibrominated analogues, we show that the mechanical strength, swelling ratio, and rate of gelation can all be controlled in a pH-sensitive manner. Moreover, we demonstrate that the degradation of pyridazinedione-gels can be induced by the addition of thiols, thus providing a route to responsive or dynamic gels, and that monobromo-pyridazinedione gels are able to support the proliferation of human cells. We anticipate that our results will provide a valuable and complementary addition to the existing toolkit of cross-linking agents, allowing researchers to tune and rationally design the properties of biomedical hydrogels.


General considerations
Proton and carbon nuclear magnetic resonance ( 1 H and 13 C NMR respectively) spectra were recorded on Bruker AvanceIII 400 (400 MHz), Avance III 600 (600 MHz), or Avance Neo 700 (700 MHz) spectrometers.NMR shifts were assigned using COSY, HSQC and HMBC spectra.All chemical shifts are quoted on the δ scale in ppm using residual solvent as the internal standard ( 1 H NMR: CDCl 3 = 7.26; DMSO-d 6 = 2.50 and 13 C NMR: CDCl 3 = 77.16,DMSO-d 6 = 39.52).Coupling constants (J) are reported in Hz with the following splitting abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, app = apparent, br = broad.Melting points (m.p.) were recorded on a Gallenkamp melting point apparatus and are uncorrected.Infrared (IR) spectra were recorded on a Perkin Elmer Spectrum 100 FTIR Spectrometer.Absorption maxima (υ max ) are reported in wavenumbers (cm -1 ).High resolution electrospray ionisation (ESI) mass spectra (HRMS) were recorded on a Waters LCT Premier XE or a Bruker Compact TOF-MS spectrometer.Nominal and exact m/z values are reported in Daltons (Da).Rheological measurements were performed on a Malvern Instruments Kinexus Pro + Rheometer fitted with a 8 mm parallel plate geometry.
Flash column chromatography was carried out with pre-loaded GraceResolv TM Silica Flash Cartridges (Grace TM ) or FlashPure EcoFlex cartridges (Büchi) on a Biotage Isolera Spektra One flash chromatography system (Biotage).Mobile phases are reported as % volume of more polar solvent in less polar solvent.Deionized water was used for chemical reactions and for protein manipulations.All other solvents were used as supplied (Analytical or HPLC grade), without prior purification.Reagents were purchased from Sigma-Aldrich, VWR, or Fluorochem and used as supplied, unless otherwise indicated.Brine refers to a saturated solution of sodium chloride.Petrol S6 refers to the fraction of petroleum ether boiling in the range 40-60 °C.Anhydrous magnesium sulfate (MgSO 4 ) was used as the drying agent after reaction workup unless otherwise stated.

Quantification of free thiols
100 µL gels were formed over 24 hrs at pH 7.4, as described above.A stock solution of Ellman's reagent (100 µL, 2 mM in pH 7.4 phosphate buffer) was then added on top, and the gels incubated at room temperature for 20 min.The supernatant was then removed and absorbance measured at 405 nm.The quantities of free thiols in the gel were then calculated based on a comparison to a standard curve of solutions of 3mercaptopropionic acid (1.6, 8, 40, 200, and 1000 µM) treated in an identical manner.

Swelling ratio calculations
Gels were formed over 24 hrs at pH 7.4, as described above, in pre-weighed 0.5 mL microcentrifuge tubes.After this time, water was added (400 µL) and the gels incubated for 2 hrs.The supernatant was then discarded and this process repeated 5 times to equilibrate the gels in pure water.The gels were then lyophilised and the dry mass measured.The gels were then swollen for 48 hrs in water (400 µL) and the swollen mass of the gels measured.The swelling ratio was calculated by dividing swollen and dry masses.Experiments were run in triplicates S14

Scanning electron microscopy
100 µL gels were formed over 24 hrs at pH 7.4, as described above.The gels were then washed with deionised water (3 × 1 h, 400 µL) and lyophilised.The samples were prepared for SEM imaging via cutting with a scalpel and affixing to an Al SEM stub via a carbon pad.
The SEM images were acquired on a JEOL JSM-7800F prime, equipped with a Schottky (field-assisted) thermionic emitter, at the York JEOL Nanocentre.An off-axis Everhart-Thornley detector with a positive bias, in LED mode 3, for attraction of both secondary and backscattered electrons was used.An objective lens aperture size of 30 μm was used with an accelerating voltage of 5 keV, resulting in a probe current of 0.1 nA.At the used working distance of 10 mm, with the aforementioned settings, the maximum resolution of the instrument is 3 nm.The images produced are 1280 x 960 pixels in resolution and were collected with a dwell time of 14 μs at each pixel.

Gel degradation by competitive thiols
After being left to form for 24 hrs, solutions of cysteamine at differing concentrations (200 µL; 0, 10, or 100 mM; 0, 0.5, or 5 equiv.thiol w.r.t.8-arm-PEG-thiol precursor) were added to the top of DiH-PD crosslinked gels.The gels were incubated for 24 hrs, and at set times were inverted to assess gel stability and photographed.

Comparison of PD-gelation strategy to previously reported vinyl sulfones
In the main manuscript we highlight that quantitative comparisons to previously reported vinyl sulfone gels are difficult due to differences in experimental set-up.
Specifically, in the prior work of Lutolf and Hubbell the gelation components are reversed with a vinyl-sulfone functionalised PEG macromer, and thiol-based crosslinker.This crosslinker is peptide-based, with N-terminal cysteine residues which are known to undergo differing reactivity to simple thiols.Moreover, gelation was performed in 0.3 M triethanolamine solutions at a desired pH, rather than phosphate buffer used in our work, which may affect gelation times and gel properties.However,

Figure S1 :
Figure S1: Plot of inverse [1MonoBr] against time following the reaction of 1MonoBr with 2 under second order conditions at a concentration of 250 µM (grey squares), and linear fit of the data (red line, R 2 = 0.99).

Figure S2 :
Figure S2: Amplitude sweep rheology measurements of gels crosslinked with bis-PDs 5 at a frequency of 5 Hz.

Figure S3 :Figure S4 :
Figure S3: Plot of swelling ratios for gels crosslinked with bis-PDs 5 in PBS after 72 hrs.

Figure S6 :
Figure S6: Plot of absolute cell numbers over time after seeding THP-1 cells on 5crosslinked hydrogels, or a tissue culture plastic control.test

Figure S7 :
Figure S7: Representative flow cytometry data of three replicates for cell viability studies of THP-1 cells cultured on 5-crosslinked hydrogels or tissue culture plastic controls.