Loading nanoliposome therapeutics into red blood cells using electroporation

We aim to prepare a novel drug delivery system by loading nanoliposome therapeutics in human red blood cells (RBCs) by electroporation, and to enhance drug circulation profiles in vivo. During our rigorous evaluation of our delivery approach, we determined that the specific type and concentration of liposomes, as well as the number of electroporation pulses used are important considerations for efficient loading of nanosystems in RBCs. We also investigated the role of a cell preservative solution on the viability of electroporated red blood cells, in an effort to increase the life span of the cells following reinjection.;Anionic liposomes were prepared using DSPC, DOPG and Cholesterol (4:5:1). The size of RBCs and liposomes was measured using 90 Plus Zeta PALS/BI-MAS. Electroporation was carried out by using high voltage pulse BTX Electrocell Manipulator 600, Genetronics. Cell viability was calculated using the hemocytometer and Trypan Blue exclusion assay. In vitro and in vivo analysis was carried out using DIC, fluorescence and intravital microscopy. Cell viability of loaded erythrocytes decreased from 100% to 59% and was maintained at 59% after 48 hours on addition of the proprietary cell protective solution. In the absence of cell protective solution, cell viability decreased from 100% to 20%. The overall morphology of the cells was preserved for 48 hours with protective solution as observed in the DIC images.;Maximum uptake by RBCs was seen at 2mumole liposome concentration. The most efficient loading of nanoliposomes occurred following the application of 4 pulses of 480Volts, 400muF capacitance and 13 ohms. The total number and overall morphology of loaded erythrocytes was maintained for 48 hours using the cell protective solution, proving it beneficial in maintaining cell viability which may translate to enhanced in vivo circulation profiles.