The retention of entrapped molecules within erythrocyte ghosts during cryopreservation

In view of the interest in erythrocyte ghosts and carrier erythrocytes as potential drug delivery systems, this work was undertaken to determine conditions facilitating the retention of entrapped molecules during cryopreservation. Upon freeze-thaw treatment intact erythrocytes and erythrocyte ghosts displayed different damage profiles with respect to cryoprotectant concentration. Non-penetrating cryoprotectants showed optimum protection of intact cells at 0.4-0.5 M; this optimum was not observed with ghosts, in which damage decreased with concentration up to 1.0 M. The concentration optimum for intact cells was not abolished by oxidative or reductive treatments suggesting that its absence in ghosts is not due to altered protein-protein or protein-lipid interactions. The extent of freeze-thaw damage to ghosts was influenced by the qualitative ionic composition of a cryoprotectant-free suspending medium, with 10-12% haemolysis observed in the presence of Li+ and Mg2+ but greater than 60% for Na+, Cs+, K+ and NH4+ with increasing loss following that order. The release on freezing of entrapped haemoglobin, insulin and sucrose was found to be inversely proportional to their molecular weights.