Bundling Process of Citrulline Polypeptides upon UCST-Type Phase Separation.

Ureido-modified poly(l-citrulline) (l-ornithine-co-l-citrulline denoted by PlOC) shows UCST-type phase separation behavior even under physiologically relevant conditions, which forms an α-helix structure above its phase separation temperature (Tp) but transforms into a solid-like aggregation composed of regular hexagonal packed cylinders below the Tp. This morphological transformation is characteristic of the phase separation behavior, but the mechanism behind it has remained incompletely understood. Here, we studied the phase separation behavior using small-angle X-ray scattering (SAXS) measurements. To analyze the SAXS data, we employed the modified unified model proposed previously, which decomposes the scattering profile into each structural element, such as the α-helices and their aggregation formed via hydrogen-bonding interactions between the ureido groups. The aggregation level is dependent on the temperature (T) and grouped into three classes: (1) mass-fractal aggregation composed of the α-helix (T > Tp), (2) spherical aggregation composed of the hexagonal packed cylinder (T < Tp), and (3) micro-order agglomeration formed by mutual fusion of the spherical aggregation, which appears as a solid-like aggregation. The SAXS analysis suggested that the transformation from the dispersed state as the α-helix to the agglomeration containing hierarchical structures occurs in a stepwise manner when the temperature falls below the Tp, which might also be transition behavior similar to the process of protein folding through folding intermediates.

Highly Ordered Polypeptide with UCST Phase Separation Behavior.

Manipulating phase separation structures of thermoresponsive polymers will enhance the usefulness of structure-controllable materials in fields such as drug delivery and tissue engineering. However, behaviors of upper critical solution temperature (UCST) have been less investigated so far, despite the importance of UCST. Here, we examined two citrulline-based polypeptides, poly(d-ornithine- co-d-citrulline) (PdOC) and poly(dl-ornithine- co-dl-citrulline) (PdlOC), to investigate how stereoregularity of the polypeptides influences UCST behavior, in addition to poly(l-ornithine- co-l-citrulline) (PlOC) previously studied. Homochiral PlOC and PdOC showed phase separation temperatures ( Tps) higher than that of racemic PdlOC. Moreover, PdlOC underwent liquid to coacervate phase separation at Tp, whereas PlOC and PdOC underwent liquid to solid-like aggregation transitions. From a structural point of view, circular dichroism and small-angle X-ray scattering measurements revealed that homochiral PlOC and PdOC polypeptides formed α-helical structures and assembled into a regular hexagonal lattice upon phase separation. Interactions between the pendent ureido groups of homochiral POCs appear to play pivotal roles in helical folding and assembly into the hexagonal structure. In addition, Tp change in response to biodegradation was confirmed for both PlOC and PdlOC. The biodegradability was considerably influenced by phase-separated structures. These findings of UCST-type POCs in this study would provide important insights into structure-controllable and thermoresponsive biomaterials.

Reversible Monolayer/Spheroid Cell Culture Switching by UCST-Type Thermoresponsive Ureido Polymers.

Multicellular spheroids have been studied in the fields of oncology, stem cell biology, and tissue engineering. In this study, we found a new polymer material for thermo-controlled spheroid/monolayer cell culture switching. The polymers that have pendant ureido groups (ureido polymers) exhibited upper critical solution temperature-type phase separation behavior. Cells in monolayer culture were converted to spheroids by the addition of ureido polymers below phase separation temperature (Tp). Time-lapse observations indicated that cells began to migrate and aggregate to form the spheroids to avoid contact with phase-separated polymer (coacervates) on the surface of the culture dish. We supposed that the coacervates seemingly suppressed interaction between cell and the dish surface or extracellular matrices. By increasing culture temperature above Tp, the spheroids began to collapse into a monolayer of cells due to dissolution of the coacervates. These results indicated that cell morphology could be repeatedly switched by changing the culture temperature in the presence of ureido polymers.