(52e) Peptoid Crosslinked Hydrogels: A Biomimetic, Synthetic Cell Culture Platform with Sequence-Defined Properties

The native extracellular matrix (ECM) is composed of hierarchically structured biopolymers containing precise monomer sequences and chain shapes to yield bioactivity. Recapitulating this structure in synthetic hydrogels is of particular interest for tissue engineering and in vitro disease models to accurately mimic biological microenvironments. However, it can be difficult to tune the properties of hydrogels precisely and independently to mimic these environments of interest. For example, the elastic modulus is frequently controlled by increasing the cross-linking density in the polymer network. This intrinsically couples hydrogel mechanics to permeability, network connectivity, and ligand density within the hydrogel system, all of which affect cell migration, proliferation, and differentiation. Thus, there is significant interest in developing a hydrogel system with tunable mechanical properties decoupled from cross-link density and network connectivity.

Toward this end, we have developed a synthetic hydrogel system using commercially available poly(ethylene glycol) (PEG) macromers with non-natural, sequence-defined poly(N-substituted glycines) (peptoids) as crosslinkers. Peptoids are of particular interest due to their large chemical diversity, tunable persistence length, and high degree of structural control. Particularly, their lack of intermolecular hydrogen bonding allows for induced secondary structure with a wide range of bulky, chiral monomers. Specifically, we have been investigating two peptoid crosslinkers: the chiral, helix inducing sequence (NsceNsceNspe)_n, where Nspe refers to (S)-N-(1-phenylethyl)glycine and Nsce is (S)-N-(1-carboxyethyl)glycine, and an analog with a racemic mixture of (S) and (R)-N-(1-phenylethyl)glycine. We hypothesized that these molecules, which will differ only in secondary structure and chain rigidity, would impart different mechanics to the hydrogel system. These peptoids were successfully synthesized via solid phase submonomer synthesis at three chain lengths with additional cysteamine-based residues included on both termini for thiol:ene crosslinking. Similarly, a peptide crosslinker was synthesized as a control. Using circular dichroism, it was determined that the secondary structure of the chiral peptoid showed significant helical character that increased with chain length. The racemic analog, however, showed significantly less helical character. Next, hydrogels were formed via thiol-norbornene photo-click chemistry using a 4-arm PEG macromer. Rheometric data indicated a marked difference in the elastic modulus of the chiral and racemic peptoid-crosslinked hydrogels. In addition, increasing the length of the chiral peptoid crosslinker increased stiffness, in direct opposition of the trend predicted by rubber elasticity theory. This method for altering stiffness utilizing polymer sequence enabled the hydrogel to maintain a constant cross-linking density and network architecture, which prompted further investigation into the utility of these novel peptoid-crosslinked hydrogels as biomimetic ECMs. We demonstrated that these hydrogels are both hydrolytically and enzymatically stable, in contrast to a peptide crosslinked control, and human dermal fibroblasts exhibit similar behavior when seeded on both peptoid- and peptide-crosslinked hydrogels functionalized with a cell adhesive ligand. Taken together, our system offers a strategy toward ECM mimics that replicate the hierarchy of biological matrices with completely synthetic, sequence-defined molecules.