Development of Icephilic ACTIVE Glycopeptides for Cryopreservation of Human Erythrocytes

Abstract

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.

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

“…Then the concentrated RBCs were lysed in DI water for the measurement of trehalose content ( c s , mg mL −1 ), or re-suspended in PBS in parallel to determine the RBC concentration (RBC, ×10 12 L −1 ) and mean corpuscular volume (MCV, ×10 −15 L) using an automated hematology analyzer (Bowlinman BM830, Beijing, China). 29 Samples incubated with 900 mOsm L −1 PBS were treated as blank ( c 0 , mg mL −1 ). The trehalose content was calculated according to the following eqn (2): 13,17,28,29,46 where 342.3 (g mol −1 ) refers to the molecular weight of trehalose and 0.7 belongs to the hRBC volume index.…”

“…29 Samples incubated with 900 mOsm L À1 PBS were treated as blank (c 0 , mg mL À1 ). The trehalose content was calculated according to the following eqn (2): 13,17,28,29,46 Intracellular trehalose mM…”

“…48 After being stored in liquid nitrogen for more than 2 h, the frozen samples were thawed in a 37 1C water bath. The cryosurvival was calculated according to the eqn (3): 17,28,29 Cryosurvival % ð Þ ¼ Cell count after freeze-thaw Cell count after incubation  100…”

“…In our previous work, hydrophobic amino acid or alkyl acid-modified e-polylysines were synthesized to promote intracellular trehalose delivery. [28][29][30] Hydrophobically modified anionic polymers were also prepared by grafting hydrophobic molecules onto the pendant carboxyl groups of g-poly(glutamic acid) (g-PGA) to facilitate intracellular trehalose delivery to sheep RBCs at 37 1C. 31 High trehalose uptake can be achieved at 37 1C by membrane fluidity due to phase transitions of the hRBC membrane at 14 1C and 34 1C.…”

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

“…31 Our previous studies demonstrated that membranedisruptive activity could be improved by phenethylamine-grafted g-poly(glutamic acid) or hydrophobic phenylalanine and lysine modified poly(aspartic acid), 18,32 while trehalose-functional and saccharide-grafted glycopeptides based on e-PL could enhance glycerol-free cryopreservation of sRBCs and hRBCs, respectively. 33,34 Based on the above achievements, in this work, we tried to synthesize new cationic polymers that could self-assemble into nanoparticles to interact with hRBC membranes. Thus, e-PL was coupled with different alkyl carboxylic acids to obtain amphipathic polymers with hydrophobic side groups, which were expected to be good candidates for delivery of trehalose into hRBCs.…”