Controlling the kinetics of thiol-maleimide Michael-type addition gelation kinetics for the generation of homogenous poly(ethylene glycol) hydrogels
Graphical abstract
To effectively study the cellular response of the material, each input parameter must be controllable. When the gel formed faster than the material could be mixed, the gelation chemically entrapped the defects created by insufficient mixing. The slowing of the gelation rate allowed for better mixing of monomers resulting in a more uniform microstructure and a more stable batch-to-batch cellular response.
Introduction
The development of poly(ethylene glycol) (PEG)-based synthetic hydrogels as an analog extracellular matrix for cell culture in vitro and tissue regeneration in vivo has been widely used and accepted [1], [2], [3], [4]. During the last decade the importance of physical properties such as, mechanical stiffness and topography of the cellular environment have been recognized to impact the cellular behavior and differentiation [5], [6], [7]. Thus, to effectively study the cellular response to the material each input parameter must be controllable and well defined. In the present study, we report the importance of balancing the gelation rate with the mixing rate for obtaining repeatable hydrogel microstructures within the widely used Michael-type addition hydrogels. We find that fast gelation kinetics lead to inefficient mixing, heterogeneous gelation, and inconsistent cell responses to the hydrogel. In particular, the fast gelation kinetics of the thiol-maleimide reaction lead to areas of low or high crosslinking that cannot be controlled unless the pH is changed to incompatible ranges. We further report on different approaches to slow down the thiol-maleimide reaction kinetics through reducing the reactivity of the thiol to result in controllable gel microstructure and cellular behavior.
Topographical and mechanical cues are strong cellular activators that can override biochemical cues [8], [9]. Thus, synthetic microenvironments aiming to generate extracellular matrix mimics must be able to control the topographical and mechanical cues throughout the material. In situ forming Michael-type addition hydrogels are typically regarded as a homogenous polymer network environment; however, in these types of hydrogels functional groups begin crosslinking immediately upon mixing (nothing is preventing the addition reaction from taking place), generating micro domains within the gels. These domains become more prominent as the reaction speed becomes greater than the mixing speed. This is a key point that has largely been ignored as the gels are assumed to be homogeneous because their bulk properties are consistent from experiment to experiment and the gel macrostructure (upon visual inspection) is uniform. Herein, we focused on demonstrating the inconsistency of fast Michael-type addition gelation and improving microstructure homogeneity of thiol-maleimide Michael-type addition reaction hydrogels by slowing the gelation kinetics of the 8-arm and 4-arm PEG-maleimide (PEG-8Mal and PEG-4Mal) hydrogels to allow for better mixing of the macromers before percolation occurs. The microstructure was compared to the fully mixed 4-arm PEG-vinylsulfone (PEG-4VS) hydrogel, a slower Michael-type addition gelation, and 8-arm PEG-Norbornene (PEG-8N) step-growth polymerized hydrogel, which is completely homogeneous, since it can be fully mixed before initializing the reaction with light. Ultimately, the thiol containing peptide fragment off either end of the bi-functional-protease-cleavable degradable linker peptide (Ac-GXCXX-GPQGIWGQ-XXCXG-NH2) (MMPXCXX) was systematically optimized for the thiolate addition to the maleimide π-bond through the modulation of its pKa.
Section snippets
PEG hydrogel formation
The PEG-8Mal (40 kDa), PEG-4Mal (20 kDa), and PEG-4VS (20 kDa) macromers were obtained from JenKem Technology USA. PEG-8N was synthesized as previously reported [10]. PEG-8Mal, PEG-4Mal, and PEG-4VS hydrogels were formed in two steps. First, the thiol-containing adhesive peptide, GCGYGRGDSPG (RGD), was reacted with the maleimide or vinylsulfone functionalized PEG-8Mal, PEG-4Mal, or PEG-4VS at a concentration of 1 mM in 100 mM HEPES pH 7.4 for 10 min (0.3 M TEOA pH 8.2 for 30 min for PEG-4VS
Results and discussion
PEG-Mal hydrogels were formed through the rapid propagation of the thiolate onto the vinyl ring of the maleimide and the sequential chain-transfer of the hydrogen (Fig. 1A) [12], [13], [14], [15], [16]. To illustrate the bulk gel properties and the microdomain generation in Michael-type addition hydrogels, we used PEG-8Mal and PEG-4Mal macromere and the widely used degradable linker peptide (Ac-GCRD-GPQGIWGQ-DRCG-NH2) (MMPCRD). Gels were formed through the rapid mixing of the PEG-Mal macromere
Conclusions
The thiolate addition to the maleimide π-bond is a faster and more efficient reaction than the thiolate addition to the acrylate π-bond. The tag sequence, CRD, optimized for the acrylate thiolate addition is no longer suitable for the new chemistry. The CRD hydrogel percolates rapidly, inhibiting mixing of the reactive monomers and resulting in variable cellular response throughout the gel. By systematically designing the tag sequence for the maleimide thiolate reaction the percolation point,
Acknowledgements
The authors would like to thank the Eugene V. Cota-Robles Fellowship for the support of this project and gracefully acknowledge that this investigation was also supported by the National Institutes of Health (NIH) under grant no. R01HL110592, R01NS079691 and under the Ruth L. Kirschstein National Research Service Award (T32-GM008185). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. Author contributions: N.J.D., Y.H. and
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