Membrane Stabilization of Poly(ethylene glycol)-b-polypeptide-g-trehalose Assists Cryopreservation of Red Blood Cells

Abstract

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.

Macromolecular cryoprotectants for the preservation of mammalian cell culture: lessons from crowding, overview and perspectives

“…An increase in the near-zero diffusion coefficient of pure water by the addition of synthetic glycopeptide (Tre) molecules prevented the diffusion of water molecules into and out of red blood cell (RBC) membranes, thereby protecting the cells during the process of cryopreservation. 71 2.1.7 Thermal stability. The glass transition temperature (T g ) of a polymeric material governs the storage temperature of the cell suspension/biospecimen to which it is added.…”

“…29 In addition, the synthetic glycopeptide (Tre) molecule demonstrated IRI activity of B45-50% (1 mg mL À1 , pH 7.4), as determined from the mean largest grain size (MLGS), owing to the presence of trehalose. 71 Apart from this, the hydrophobic derivatives of polyampholyte-based cryoprotective polymers also showed IRI activity, preventing cell death. 76 The use of polymeric crowders (for example, Ficoll s , dextran, etc.)…”

“…86 In addition, the presence of glycopeptides in the synthetic glycopeptide (Tre) molecules enhanced the stabilization of RBC membranes, as evident from their low hemolytic activities, the interactions energies calculated using molecular dynamics simulation, and water diffusion coefficients determined at À196 1C. 71 Besides this, Jain et al demonstrated that polyampholytes are adsorbed on cell membranes during the freezing process and their cryoprotective mechanism was thought to be due to membrane protection. 87 2.1.13 Alteration of membrane transition temperature.…”

“…29 Synthetic glycopeptide(Tre) developed for cryopreservation of RBCs exhibited hydrophilic, hydrophobic, and ampholytic properties owing to the presence of neutral and hydrophilic PEG and trehalose, cationic lysine, anionic aspartic acid, and hydrophobic phenylalanine moieties in its structure. 71 Additionally, poly(methyl glycidyl sulfoxide) (PMGS), a polymeric macromolecule with DMSO-like functional properties, was synthesized and evaluated for its cryoprotective potential. 66 Polyampholytes, another class of macromolecules belonging to this category, bear both positive and negative charges.…”

“…Weakly acidic pH (pH = 6) resulted in higher cryo-survival of RBCs while higher concentrations of synthetic glycopeptide-Tre resulted in lower values of diffusion coefficients of water suggesting better cell membrane protection at À196 1C. 71 Apart from this, the water, salt and polymer chain dynamics play important roles in improving cryopreservation using polyampholytes. It was observed that higher osmotic pressure in the freezing medium containing polyampholytes caused lower post-thaw cell viability.…”