Combination of hydrophobically modified γ-poly(glutamic acid)and trehalose achieving high cryosurvival of RBCs

Trehalose is expected to be an alternative for toxic glycerol as a biocompatible cryoprotectant of red blood cells(RBCs).In this work,γ-poly(glutamic acid)(PGA)is modified by grafting hydrophobic phenethylamine,3,4-dimethoxyphenylethylamine and dodecylamine,respectively.The graft-modified PGA can significantly enhance cryosurvival of RBCs in combination with trehalose.Analyses of dynamic light scattering,hemolysis assay,atomic force microscope and confocal laser scanning microscope suggest that the modified PGA polymers can self-assemble into nanoparticles in phosphate buffer saline solutions at the pH range of 6.0–7.4,and exhibit membrane-disruptive activity due to hydrogen bond,conjugation and hydrophobic interactions with cell membranes.It is assumed that the modified PGA polymers can improve the cryosurvival of RBCs by promoting membrane permeability of trehalose.Among the three graft-modified polymers,phenethylamine-grafted PGA(PGA-g-PEA)can significantly increase the intracellular trehalose-loading content to 11.3±2.4 m M at pH 7.4,much higher than the value0.17±0.66 m M when trehalose is used without any polymers.In view of the aforementioned merit,the cryosurvival rate of sheep RBCs is increased to about 90%by incubation with 1.0 mg mL-1 PGA-g-PEA and 0.36 M trehalose.In vitro cell culture of L929 fibroblasts demonstrates low cytotoxicity of PGA-g-PEA.Therefore,hydrophobic PEA-modified PGA with enhanced intracellular trehalose-loading ability can be potentially applied in glycerol-free RBC cryopreservation or other related biomacromolecule delivery systems.