57. A transmembrane mega highway for cryoprotective agent delivery via self-assembling organic nanopores with tunable function

Abstract

Cryoprotective agents (CPAs) are critical additives that improve the post-thaw viability of cryopreserved biological systems by preventing ice crystal nucleation and growth. Membrane permeable CPAs also prevent osmotic shrinkage of the cells and reduce the volume of available water by penetrating and equilibrating across the cell membrane. All known CPAs exhibit various levels of cytotoxicity at their effective concentration which may be decreased by reducing the CPA loading temperature and exposure time. However, most CPAs become effectively impermeable at sub-zero temperatures. This is largely associated with the dysfunction of transmembrane protein channels at low temperature that serve as mass transport gates. An unbalanced gradient of CPA and salt across cell membrane leads to detrimental cell shrinkage as water efflux out of the cell. This could be detrimental to the fate of large, complex solid tissues/organs by damaging cell–cell and cell–matrix junctions. To facilitate the intracellular delivery and transmembrane equilibration of the CPA at sub-zero temperatures, we propose size and function tunable organic nanopores as a “mega highway” to deliver CPAs across cell membrane which allows a significant decrease in both the CPA exposure time and temperature during freezing. This method may be particularly effective when CPA loading in tissues using the “liquidus tracking” or step-wise methods where increasingly concentrated solutions of CPA are loaded in the tissue/organ at progressively decreasing temperatures. Recent work in Gong’s group has demonstrated the reliability of constructing well-defined nanotubular assemblies via the enforced stacking of shape-persistent macrocycles based on the interplay of multiple hydrogen-bonding, dipole-dipole, and aromatic π - π stacking interactions. The resultant nanotubes have modifiable surfaces and non-collapsible inner pores of a uniform diameter defined by the constituent macrocycles. The robust self-assembling nanopores have been found to act as transmembrane channels which can mediate highly selective transmembrane ion transport with high stability, unprecedented for synthetic ion channels, as well as exhibiting highly efficient transmembrane water permeability, which is comparable to aquaporin. Aquaporins have been found to efficiently assist transport of water and glycerol during freezing to prevent cell rupture under osmotic pressure. In this proposal, selected organic nanopores are delivered into a target system at physiological temperature (37 °C), followed by CPA loading at hypothermic temperature (e.g. 1 °C). A high influx rate of CPAs through shape-persistent nanopores can be maintained during cooling as a function of the concentration gradient across cell membrane, thereby reducing the required time to reach vitrification concentrations. Upon rewarming, these organic nanopores will facilitate rapid removal of CPAs to reduce exposure time and the consequent toxic side effects. The proposed incorporation of synthetic nanopores can significantly reduce toxicity and cell injury due to osmotic shrinkage caused by CPAs and salt during both the cooling and rewarming processes via (1) reducing CPA exposure time and (2) enabling rapid CPA loading and unloading at lower temperatures. Moreover, versatile functional organic nanotubes of diverse sizes and properties by modifying inner macrocyclic cavities allow selective CPA transport by while preventing ion exchange. Incorporation of functional supramolecular assemblies to enhance membrane permeability of CPAs could lead to a revolutionary solution to cryopreserve complex biological systems.