Methological Evaluation of the Hypotonic Dialysis Method for Encapsulation of Drugs in Erythrocytes

Hypo-osmotic (hypotonic) dialysis method is a gentle osmotic-based method for encapsulation of a wide variety of drugs in erythrocytes, producing carrier cells with apparently normal shapes and functions. A simple and available method has been developed and evaluated for preparation of long-circulating carrier erythrocytes. A series of primary methodological variables, including initial hematocrit, tonicity and volume ratios used in the hemolysis, restoration and re annealing steps, and duration of each step were evaluated in terms of their relative impacts on the overall recovery of the developed carrier cells. It was shown that the very simple method developed on hypo-osmotic dialysis platform is a robust method in terms of the effect of changing parameter conditions, with the cell lysis index being 15 percent in the worst cases. Based on the results, an initial hematocrit of 75%, a sac-to-dialysate volume ratio of 1/100 in all three stages, a tonicity of hypotonic medium with no limitation, a hypertonic medium with tonicity of 2 times the eutonic buffer, with the duration of the hypotonic stage being critical, thus should be taken cautiously based on the specific drug to be loaded, but the duration of the isotonic and hypotonic stages being not critical within the time range tested, were selected collectively as the optimum condition. The validation tests on the optimum condition were indicative of the validity of the selected point estimates.