S T R U C T U R E O F M A T E R I A L S

S C I E N T I F I C H I G H L I G H T S

1 3 8 H I G H L I G H T S 2 0 2 2 I

Fig. 130: Ion-triggered hydrogels self-assembled from statistical copolypeptides.

In-situ SAXS analyses of gelation mechanism of hydrogels

Through one-step, ring-opening polymerisation, statistical copolypeptides comprising lysine and tyrosine were obtained. This polymer can form a soft hydrogel through adding salt ions. To understand this gelation mechanism, in-situ X-ray absorption analyses were performed, revealing that the gelation of these polypeptides is due to the formation of intermolecular β-sheet secondary structures.

Hydrogels comprise three-dimensional crosslinked networks allowing them to hold significant amounts of water, which makes them attractive materials for biomedical applications such as drug delivery, wound dressing, antimicrobial coatings and regenerative medicine. Natural hydrogels based on proteins (e.g., collagen, gelatine, fibroin) or carbohydrates (e.g., alginate) have long been the materials of choice due to their inherent biocompatibility. These materials form physical hydrogels through peptide self-assembly or ion interaction in alginates. Although these natural polymers have drawn large attention from the biomaterials research community, the difficulty in their purification as well as the lack of structural control has motivated researchers to explore synthetic analogues. One approach to obtain synthetic polypeptide hydrogels is using (multi)block copolypeptides, in which the design principle is always focused on amphiphilic block structures. The ability of these block copolypeptides to form three-dimensional networks in an aqueous medium relies solely on their self-assembly through hydrophobic interactions. Sequential amino acid N-carboxyanhydride (NCA) polymerisation has been employed to produce well-defined hydrogellating block copolypeptides. This includes linear as well as branched block copolypeptides, comprising mainly lysine or glutamic

acid in their hydrophilic blocks and amino acids such as phenylalanine, isoleucine, leucine or alanine in their hydrophobic blocks.

To further simplify the synthesis of polypeptide hydrogelators, it would be desirable to revert to a one- step process of statistical copolymerisation. However, statistical copolypeptides have not been proposed as hydrogelators to date. As these structures have no defined (neither in length nor sequence) hydrophobic domains suitable for self-assembly, it is counterintuitive to assume their hydrogelation. That is, unless a different mechanism not relying on the hydrophobic phase separation of conventional amphiphilic block copolypeptides can be found for physical crosslinking. Ideally, the hydrogelation of statistical copolypeptides can be externally triggered by salting-out , as occurs in natural proteins. Notably, to the best of our knowledge, hydrogelation in salt solution has never been considered as a gelation mechanism for amphiphilic block copolypeptides, owing to the destructive impact salts have on amphiphilic interactions, but can be achieved with sequence-defined oligopeptides or homopolypeptides. This study reports for the first time a statistical copolypeptide comprising lysine and tyrosine (Figure 130) that is capable of forming hydrogels upon addition of buffer salt solution.

To obtain more insights into the morphological changes during the gelation process, a series of in- situ small-angle X-ray absorption spectroscopy (SAXS) analyses were carried out on the hydrogels at beamline BM26 (DUBBLE). Here, a flexible worm-like polymer chain model was used to fit the system, as suggested by previous studies on peptide-based self-assembly systems. As shown in Figure 131a, for samples containing P(lys80-tyr20) mixed with 0.1 M PBS buffer, all scattering profiles can be fitted with a combination of