A Dynamic Membrane-Active Glycopeptide for Enhanced Protection of Human Red Blood Cells against Freeze-Stress.

Intracellular delivery of freezing-tolerant trehalose is crucial for cryopreservation of red blood cells (RBCs) and previous strategies based on membrane-disruptive activity usually generate severe hemolysis. Herein, a dynamic membrane-active glycopeptide is developed by grafting with 25% maltotriose and 50% p-benzyl alcohol for the first time to effectively facilitate entry of membrane-impermeable trehalose in human RBCs with low hemolysis. Results of the mechanism acting on cell membranes suggest that reversible adsorption of such benzyl alcohol-grafted glycopeptide on cell surfaces upon weak perturbation with phospholipids and dynamic transition toward membrane stabilization are essential for keeping cellular biofunctions. Furthermore, the functionalized glycopeptide is indicative of typical α-helical/ß-sheet structure-driven regulations of ice crystals during freeze-thaw, thereby strongly promoting efficient cryopreservation. Such all-in-one glycopeptide enables achieving both high cell recovery post-thaw >85% and exceptional cryosurvival >95% in direct freezing protocols. The rationally designed benzyl alcohol-modified glycopeptide permits the development of a competent platform with high generality for protection of blood cells against freeze-stress.

Membrane stabilization versus perturbation by aromatic monoamine-modified γ-PGA for cryopreservation of human RBCs with high intracellular trehalose.

As a nonreducing disaccharide, trehalose can be used as a biocompatible cryoprotectant for solvent-free cell cryopreservation, but the membrane-impermeability limits its cryoprotective efficiency. Herein, a series of aromatic monoamines with a 1-4 methylene spacer were grafted onto γ-poly(glutamic acid) (γ-PGA) for promoting intracellular trehalose uptake in human red blood cells (hRBCs) via membrane perturbation. The self-assembled nanoparticles of the obtained amphiphilic γ-PGA could be adsorbed on the cell membrane by the hydrophobic interaction to disturb the lipid arrangement and increase the membrane permeability of trehalose under hypertonic conditions. Results suggested that the intracellular trehalose could be enhanced progressively with the methylene spacer length, significantly increasing to 75.1 ± 0.7 mM by incubating hRBCs in 0.8 M trehalose containing phenylbutylamine-grafted γ-PGA at 4 °C for 24 h. Meanwhile, the other three polymers exhibited membrane stabilization in addition to improved intracellular trehalose, maintaining the membrane integrity during cryopreservation to achieve high cryosurvival. Molecular dynamics simulation further confirmed that defects could be formed by interaction of the above four amphiphilic polymers on the modeled phospholipid bilayer. It was believed that glycerol-free cryopreservation of human cells could be realized by using trehalose as the biocompatible cryoprotectant, and membrane stabilization can be a compensatory approach to membrane perturbation during impermeable biomolecule delivery.

NonFreezable Preservation of Human Red Blood Cells at -8 °C.

Red blood cell (RBC) preservation is very important in human health. The RBCs are usually preserved at 4 ± 2 °C without freezing or at a very low temperature (-80 °C or liquid nitrogen) with deep freezing. Herein, non freezable preservation of RBCs at a subzero temperature is reported to prolong the preservation time compared with that at 4 ± 2 °C. By adding glycerol and poly(ethylene glycol) (PEG) (average number molecular weight 400, PEG-400) into the preservation solution, the freezing point is decreased and the hemolysis is kept low. The cell metabolism of stored RBCs at -8 °C is reduced, and the shelf life of RBCs extends up to at least 70 days. At the end of preservation, the pH decreases a little bit to demonstrate the low metabolic rate of RBCs stored at subzero temperatures. After quick washing, the RBC survival rate is ca. 95%. The adenosine triphosphate, 2,3-diphosphoglycerate, and cell deformation ability of the washed RBCs are maintained at a high level, while the malondialdehyde is relatively low, which verifies the high quality of RBCs stored at this condition.

Cryopreservation of human erythrocytes through high intracellular trehalose with membrane stabilization of maltotriose-grafted ε-poly(L-lysine).

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.

Facilitating trehalose entry into hRBCs at 4 °C by alkylated ε-poly(L-lysine) for glycerol-free cryopreservation.

Currently, glycerol is a conventional cryoprotectant of human red blood cells (hRBCs), but the time-consuming thawing and deglycerolization processes are essential before transfusion. Much of the research up to now has been conducted on the delivery of impermeable trehalose to hRBCs at 37 °C, but the cryoprotective effect of trehalose and deterioration of cells still remain challenging. Encouraged by the interaction of hydrophobic or cationic groups on cell membranes and osmotic stabilization, herein, we propose a novel cryopreservation system to facilitate trehalose entry into hRBCs at 4 °C and pH 7.4. High intracellular trehalose contents and cryosurvival of hRBCs were achieved with small function variations via the assistance of self-assembled nanoparticles of alkylated ε-poly(L-lysine) (ε-PL) along with poly(vinyl pyrrolidone) (PVP). The effect of amphipathic alkylated ε-PL with various alkyl chains and grafting ratios on membrane perturbation with protection of PVP was systematically investigated. Overall, by the combination of alkylated ε-PL and PVP, the intracellular trehalose could be enhanced to 109.7 ± 6.1 mM and subsequently hRBC cryosurvival reached 91.7 ± 5.5%, significantly higher than those containing trehalose only, 11.9 ± 1.1 mM and 50.0 ± 2.1%, respectively. It was observed that the biocompatible trehalose-loading system could benefit glycerol-free cryopreservation of hRBCs and also provide a feasible way for impermeable biomacromolecule delivery.

Development of Icephilic ACTIVE Glycopeptides for Cryopreservation of Human Erythrocytes.

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.