Loading Erythrocytes with Insulin and Determining the Intraerythrocytic Concentration and Stability.

Abstract

Background: Novel insulin delivery methods (IDMs) are needed to evade the inconveniences of current IDMs. Objective: To load soluble insulin (SI) into human erythrocytes and determine the intraerythrocytic concentration using UV absorptions and the stability of insulin-loaded erythrocytes (ILEs) with time Methods: A calibration curve for insulin was developed using UV absorption data from a standard dilution series of SI to develop a computerized application (CA) to analyze SI. Blood samples were obtained from healthy volunteers aged 25 to 55 years. Erythrocytes were isolated from the whole blood to identify the optimum NaCl concentration for opening erythrocytes. The erythrocytes were closed after transferring SI. The transferred and loaded SI were determined using the CA. The loading capacity was measured at 10, 20, and 30 minutes. The stability of ILEs was visually assessed every 24 hours since the loading process using a trinocular microscope at 400x magnification. The One-way ANOVA test was done using SPSS version 16.0 to compare the mean values. Results: The SI showed good liner agreement in the range of 0.2 to 0.8 absorbance at 276 nm (r=0.998), proving a simple and affordable CA. The optimum concentration of NaCl solution was 0.7% w/v to open the erythrocytes, and the optimum concentration of 20 IU/mL SI solution was transferred into the erythrocytes. It was shown that 16.28±0.19 IU, 18.56±0.17 IU, and 19.19±0.05 IU of insulin were transferred into 1.00 mL of erythrocytes after 10, 20, and 30 minutes (p<0.05), respectively. An insulin amount of 6.96± 0.17 IU, 7.27±0.24 IU, and 7.69±0.49 IU related to 10, 20, and 30 minutes of exposure (p>0.05) had loaded into 1.00 mL of erythrocytes. The stability period for ILEs was 72 hours. Conclusions: The hypo-osmolarity-based insulin loading process successfully loaded enough insulin into erythrocytes. The ILEs have a three-day stability period in a 0.9% NaCl solution under 20-80 C storage conditions.