Table 3:
Summary of studies done on lyophilized erythrocytes
| Results | ||
|---|---|---|
| Evaluating stability of lyophilized lipid encapsulated hemoglobin (LEH) | Stability of frozen LEH, lyophilized LEH and lyophilized LEH containing trehalose as excipient was compared over a time of 6 months | For lyophilized LEH particle size increased during rehydration, and hemoglobin content decreased, while size for formulation with trehalose remained unchanged, and initially slight hemoglobin oxidation was observed but it remained unchanged over time. [78] |
| Effect on metabolic activity of erythrocytes due to lyophilization | The activity of hemoglobin is regulated by ATP and 2,3-diphosphoglycerate, produced during glycolysis through Embden-Meyerhof pathway and pentose-phosphate pathway. Whether any of the enzymes in these pathways were affected was studied. Results were compared against that of fresh non-lyophilized erythrocytes. | A part of glycolytic enzymes remains unaffected, while 3 enzymes decreased significantly in lyophilized erythrocytes. Lyophilized erythrocytes had unaffected Methemoglobin reduction pathway, hence usual oxygen transport would not be hampered, and even with slight alterations in enzymes produced, it can be considered as a viable option. [26] |
| Study on determining effective freezing rate for freeze drying erythrocytes | The recovery rate for hemoglobin and stability were determined and compared against thawed erythrocytes | Cooling rate of 200K/min was found to be effective and as cooling rate was increased before lyophilization, recovery rate for hemoglobin also increased, suggesting higher freezing rate leads to better cryopreservation. While for thawed sample increasing cooling rate lead to lower stability. [27] |
| Evaluating effect of freeze drying of erythrocytes at ultra-low temperature | As cryoprotectants, hydroxyethyl starch and disaccharide D-maltose was used and temperature for −5 to −65°C was used. | Highest stability, i.e. Recovery rate for hemoglobin, after resuspension was observed at −35°C and the authors suggest the reason is higher temperature leading to damaging effects and colder temperature leading to insufficient driving force for water transport. [28] |
| Preservation of erythrocyte activity with trehalose | Formulation prepared with trehalose in presence of hydroxyethyl starch, and human serum albumin. Rate of hemolysis, methemoglobin formation and metabolites of glycolysis were tracked in the lyophilized RBC | Secondary structure of hemoglobin and major metabolic pathways remain unchanged, with 50% hemolysis during rehydration. Hence trehalose loaded formulations could be stable lyophilized erythrocytes. [29] |
| Using phospholipids with trehalose for freeze drying erythrocytes | Phospholipids were hypothesized to protect membrane from damage during drying. Hemolysis (%) and methemoglobin level were compared in formulations with trehalose, with trehalose and phospholipid, with phospholipids only and for cases without either trehalose or phospholipid. | Trehalose and phospholipid loaded erythrocytes had lowest amount of methemoglobin formation and least percent hemolysis, suggesting a stable formulation for lyophilized erythrocytes. [79] |
| Determining stability of lyophilized hemoglobin for DCIP test | Lyophilized hemoglobin and variant hemoglobin E were used in the test and storage duration of 12 months was used | Lyophilized hemoglobin could be used as reliable control in the test due to its stability over the storage duration [80] |