Nω-(4-Toluenesulfonyl)-L-arginine

Ice formation and recrystallization exert severe impairments to cellular cryopreservation. In light of cell-damaging washing procedures in the current glycerol approach, many researches have been devoted to the development of biocompatible cryoprotectants for optimal bioprotection of human erythrocytes. Herein, we develop a novel ACTIVE glycopeptide of saccharide-grafted ε-poly(L-lysine), that can be credited with adsorption on membrane surfaces, cryopreservation with trehalose, and icephilicity for validity of human erythrocytes. Then, by Borch reductive amination or amidation, glucose, lactose, maltose, maltotriose, or trehalose was tethered to ε-polylysine. The synthesized ACTIVE glycopeptides with intrinsic icephilicity could localize on the membrane surface of human erythrocytes and improve cryopreservation with trehalose, so that remarkable post-thaw cryosurvival of human erythrocytes was achieved with a slight variation in cell morphology and functions. Human erythrocytes (~50% hematocrit) in cryostores could maintain high cryosurvival above 74%, even after plunged in liquid nitrogen for 6 months. Analyses of differential scanning calorimetry, Raman spectroscopy, and dynamic ice shaping suggested that this cryopreservation protocol combined with the ACTIVE glycopeptide and trehalose could enhance the hydrogen bond network in nonfrozen solutions, resulting in inhibition of recrystallization and growth of ice. Therefore, the ACTIVE glycopeptide can be applied as a trehalose-associated "chaperone", providing a new way to serve as a candidate in glycerol-free human erythrocyte cryopreservation.

Cryopreservation of human erythrocytes via suitable cryoprotectants is essential for transfusion during emergencies, but the conventional glycerolization method requires a tedious thawing-deglycerolization process. Alternatively, trehalose, a nonreducing disaccharide, has gained much attention as a biocompatible cryoprotectant due to its nature in living organisms capable of surviving extreme cold and desiccation. In this work, cryopreservation of human erythrocytes was realized through high intracellular trehalose enhanced by benzyl alcohol at 4 °C with membrane stabilization of maltotriose-grafted ε-poly(L-lysine). Intracellular trehalose could reach 94.2 ± 12.1 mM with slight impacts on morphology and cell functions, and the post-storage cryosurvival of human erythrocytes could achieve 96.2 ± 3.4% via membrane protection by the glycopeptide. It has been demonstrated that the functional glycopeptide performed as an extracellular cryoprotectant accompanied by high intracellular trehalose for synergistic cryopreservation of human erythrocytes in the biocompatible glycerol-free conditions. This two-step approach involving augmentation of intracellular trehalose at a hypothermic temperature and membrane stabilization of the functional glycopeptide could be an alternative way for human cell cryopreservation.

As alternative cryoprotectants of conventional organic glycerol, biocompatible synthetic glycopeptides that can assist cryopreservation of red blood cells (RBCs) are proposed in this study. A series of glycopeptides are synthesized via ring-opening polymerization of N-carboxyanhydrides of lysine, aspartic acid, and phenylalanine initiated by poly(ethylene glycol)-NH2 and followed by chemical tethering carboxylated trehalose to the pendant amino moieties. The synthetic glycopeptides demonstrate distinguished features of low cytotoxicity, low hemolysis, and cell membrane stabilization. The specifically designed glycopeptides enhance cryosurvival recovery of sheep RBCs up to 87.3 ± 0.3% at pH 6.0 and 86.5 ± 0.3% at pH 7.4 together with trehalose during cryopreservation. The synergistic cryopreservation of RBCs is achieved via membrane stabilization of the glycopeptide and ice recrystallization inhibition of trehalose during freezing and thawing. Molecular dynamics simulation indicates that the glycopeptides enhance membrane stabilization at -196 °C by tuning water diffusion. This work provides a potential option of highly efficient and glycerol-free RBC cryopreservation by combination of synthetic glycopeptides and trehalose, which would inspire design and utilization of different mechanisms for the effective cryopreservation of cells or tissues.