Degradable and Multifunctional PEG-Based Hydrogels Formed by iEDDA Click Chemistry with Stable Click-Induced Supramolecular Interactions

Abstract

The inverse electron demand Diels-Alder (iEDDA) reactions are highly efficient click chemistry increasingly utilized in bioconjugation, live cell labeling, and the synthesis and modification of biomaterials. iEDDA click reactions have also been used to cross-link tetrazine (Tz) and norbornene (NB) modified macromers [e.g., multiarm poly(ethylene glycol) or PEG]. In these hydrogels, Tz-NB adducts exhibit stable supramolecular interactions with a high hydrolytic stability. Toward engineering a new class of PEG-based click hydrogels with highly adaptable properties, we previously reported a new group of NB-derivatized PEG macromers via reacting hydroxyl-terminated PEG with carbic anhydride (CA). In this work, we show that hydrogels cross-linked by PEGNB_CA or its derivatives exhibited fast and tunable hydrolytic degradation. Here, we show that PEGNB_CA (either mono- or octafunctional) and its dopamine or tyramine conjugated derivatives (i.e., PEGNB-D and PEGNB-T) readily cross-link with 4-arm PEG-Tz to form a novel class of multifunctional iEDDA click hydrogels. Through modularly adjusting the macromers with unstable and stable iEDDA click-induced supramolecular interactions (iEDDA-CSI), we achieved highly tunable degradation, with full degradation in less than 2 weeks to over two months. We also show that secondary enzymatic reactions could dynamically stiffen these hydrogels. These hydrogels could also be spatiotemporally photopatterned through visible light-initiated photochemistry. Finally, the iEDDA-CSI hydrogels post ester hydrolysis displayed shear-thinning and self-healing properties, enabling injectable delivery.

Figure 1

Overview of synthesis and cross-linking of PEGNB, PEGNB_CA, and PEGNB-X macromers via thiol-norbornene or tetrazine-norbornene reactions. (A) Schematic of norbornene functionalization onto PEG-hydroxyl through either Steglich esterification with norbornene acid or ring-opening ester conjugation using carbic anhydride, yielding (m)PEG(8)NB and (m)PEG(8)NB_CA, respectively. PEGNB_CA can be further conjugated with different amines, yielding (m)PEG(8)NB-X (i.e., (m)PEG(8)NB-D, (m)PEG(8)NB-T, and (m)PEG(8)NB-I). (B) Cross-linking (m)PEG(8)NB_CA or (m)PEG(8)NB-X with PEG4SH via photoinitiated thiol–ene reaction (top) or with PEG4Tz via spontaneous iEDDA reaction (bottom). (C) In situ photorheometry of PEG8NB-D and mPEGNB-D with PEG4SH in the presence of 2 mM LAP and 365 nm light exposure at 19 mW/cm² intensity. (D) In situ rheometry of PEG8NB-D and mPEGNB-D with PEG4Tz at 37 °C for 30 min. (E) Pictures of hydrogel precursor solution containing 5 wt % PEG8NB-D with PEG4SH and 2 mM LAP or with PEG4Tz at R([Tz]/[NB]) = 1 before cross-linking, posthydrogel cross-linking (i.e., 2 min 365 nm exposure at 3 mW/cm² for thiol–ene or 1 h incubation at 37 °C for tetrazine-ene), and after 24 h of incubation in pH 7.4 PBS at 37 °C. Thiol–ene hydrogels completely degraded, while iEDDA hydrogels were structurally intact after 1 day.

Figure 2

Cross-linking and degradation of hydrogels formed by variants of PEG-norbornene. (A) Reaction rate constants of PEG8NB, PEG8NB_CA, and PEG8NB-D as determined by photospectrometry. (B) Starting storage moduli (G′) of 5 wt % 8-arm PEG8NB-D hydrogels cross-linked with either PEG4SH (thiol-norbornene reaction) or PEG4Tz (tetrazine-norbornene reaction). Reactive group stoichiometric ratios (R = [Tz]/[NB]) were set at 1. (C) Ester hydrolysis and degradation of iEDDA hydrogels cross-linked by PEG8NB-D with either PEG4SH (thiol-norbornene reaction) or PEG4Tz (tetrazine-norbornene reaction). Changes in G′ were normalized to the starting modulus over a 24 h period. Curve fitting represents best-fit pseudo-first-order kinetics of ester hydrolysis. (D) ¹H NMR spectra of PEG8NB-D, PEG8OH, and leaving degradation products collected through dialysis. (E) ATR-FTIR spectra of PEG8NB-D iEDDA click cross-linked hydrogels before and after hydrolytic degradation.

Figure 3

Characterization of iEDDA click hydrogel cross-linking and degradation. (A–C) Changes in G′ of iEDDA hydrogels cross-linked by PEG4Tz and PEG8NB (A), PEG8NB_CA (B), and PEG8NB-D (C). (D) Starting G′ of PEG4Tz/mPEGNB-D hydrogels cross-linked at different concentrations of [Tz-NB-D]. (E) Changes in G′ of mPEGNB-D iEDDA-CSI hydrogels at different [Tz-NB-D]. (F) Changes in G′ of mPEGNB-D hydrogels compared to mPEGNB_CA and mPEGNB. (G) Tuning the degradation behavior through different mPEGNB-D/mPEGNB_CA blends at different total [Tz-NB] concentrations. A higher [NB-D] leads to a higher iEDDA-CSI that stabilizes the hydrogel, while a higher [NB_CA] disrupts the supramolecular interactions between the Tz-NB adducts and weakens the hydrogel.

Figure 4

Cytocompatibility of iEDDA-CSI hydrogels. (A) Confocal images of hiPSCs encapsulated within 3.75 wt % PEG8NB-T cross-linked with 4-arm PEGTz. Cells were stained with calcein AM and ethidium homodimer at different time points. (B) Average spheroid size determined from ImageJ using live/dead confocal images at different time points. (C) Confocal images of hiPSCs stained with NANOG, OCT4, and SOX2. Cells were counterstained with DAPI after 7 days of culture. (D) Confocal image of lumen formation of hiPSC spheroid on day 7. Cells were stained with F-actin and counterstained with DAPI.

Figure 5

Enzymatic stiffening and photopatterning of (m)PEGNB-T iEDDA-CSI click cross-linked hydrogels. (A) Schematics of dityrosine cross-linking catalyzed by mushroom tyrosinase and visible light-mediated ligand conjugation using FMN of FITC-tyramide. (B) Storage moduli of mPEGNB-T iEDDA-CSI hydrogels on days 0 and day 2. (C) Stiffening of mPEGNB-T iEDDA-CSI hydrogels using MT at different concentrations over an 8 hour period with corresponding normalized G′ values. (D) Confocal image of a PEG4Tz/PEG8NB-T iEDDA hydrogel containing photopatterned lines using FITC-tyramide conjugation via FMN and visible light-mediated dityrosine cross-linking. Light intensity across the photopatterns (dashed line) was analyzed by the histogram feature in ImageJ.

Figure 6

Assessing shear thinning and self-healing properties of iEDDA click cross-linked PEGNB-D hydrogels post ester hydrolysis. (A) Measurement of viscosity at different shear rates of PEG4Tz/PEG8NB-D hydrogels. (B) Gel–sol transition of PEG4Tz/PEG8NB-D hydrogels under increased shear strain. (C) Gel–sol–gel transition based upon alternating between high to low shear strain over time. (D) Pictures of extruding loaded PEG4Tz/PEG8NB-D hydrogels from a syringe through an 18G needle.