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Modulating ice nucleation and growth with bio-inspired protein-polymers for cryopreservation

Periodic Reporting for period 2 - PROTECT (Modulating ice nucleation and growth with bio-inspired protein-polymers for cryopreservation)

Reporting period: 2023-01-01 to 2024-06-30

Efficient, non-toxic cryoprotectants, that allow long-term storage of viable therapeutic cells and tissues, are the tool that regenerative medicine requires for its successful realization into a viable therapeutic option. A remarkable class of biopolymers known as ice-binding proteins (IBPs) alleviate the risk of freeze injury throughout the Kingdoms of Life by keeping the nucleation and growth of ice crystals in check. Yet, the application potential of IBP analogues (IBPAs) as cryoprotectants has remained underexploited. This is because we are yet to unravel and utilize the structure-function relations, which govern the activity of IBPAs as inhibitors of ice recrystallization and promoters of ice nucleation at the single-molecule level in vitro and within a complex biological environment. PROTECT aims to develop uniquely modular IBPAs to perform the quantitative single-molecule and physico-chemical experiments essential to bridge this knowledge gap and to engineer ice-binders optimized for cryopreservation. Our first aim is the biosynthesis of a novel class of ice-binding protein-polymers (iPP) with systematic variations in composition and size. Ice nucleators with a broad range of sizes will be created from iPPs of variable chain length by dissolution, self-assembly and surface-tethering to nanoparticles of variable dimensions. Super-resolution microscopy experiments of iPP ice-binding will deliver high-resolution maps of spatiotemporal distribution and dynamics. These will be related to iPP structure, physico-chemical properties, ice recrystallization inhibition (IRI) and ice nucleation (IN) activity. These insights will translate into the next generation of bioactive iPPs tailored to maximize both IRI and IN. Their impact on heart cell and tissue cryopreservation will be examined to advance our fundamental understanding of freeze injury and dramatically improve post-thaw recovery as well as structural and functional integrity without adverse effects.
We have performed the first super-resolution microscopy experiments on ice-bound polypeptides, which will result in high-resolution maps of spatiotemporal distribution and dynamics.
We have reported the first de novo designed ice-binding proteins. This series of computationally designed ice-binding polypeptides has helped to advance our understanding of structure-activity relations and, moreover, they are much more stable than their natural counterparts.
We are performing the world's first super-resolution microscopy experiments on novel ice-interactive materials. These state-of-the-art on-ice imaging experiments will result in high-resolution maps of the spatiotemporal distribution and dynamics of novel ice-binders. This will advance insight in the structure-activity relations of the novel ice-binders.