Enzyme loading of electrically homogeneous human red blood cell ghosts prepared by dielelctric breakdown

Human red blood cell ghosts were prepared by electrical haemolysis at 0 degrees C in isotonic solutions using a discharge chamber which was part of a high voltage circuit. The size distribution of the ghosts was normally distributed, the modal (=mean) volume was approx. 115 mum3, performing the electrical haemolysis in the following solution: 105 mM KCI, 20 mM NaCL, 4mM MgCl2, 7.6 mM Na2HPO4, 2.94 mM NaH2PO4, 10 mM glucose, pH 7.2. Resealing was carried out at o degrees C for 10 min (after the haemolytic step) and then for further 20 min at 37 degrees C. The mean volume of the ghost preparation could be changed by variation of the phosphate concentration in the above solution replacing a part of NaCl by phosphate (5 mM phosphate: 94 mum3, 15 mM phosphate: 135 mum3). The breakdown voltage of the ghost cell membranes measured with a hydrodynamic focusing Coulter Counter depends on the mean volume (94 mum3 = 1.04 V, 134 mum3 = 1.36 V). On the other hand, the breakdown voltage is constant throughout each size distribution pointing to an "electrically homogeneous" ghost preparation. The sensitiviity of the Coulter Counter to detect electrical inhomogeneities in the membranes of a ghost population is demonstrated by dielectric breakdown measurements of an apparently normally distributed ghost preparation containing two different "electrically homogeneous" ghost population i.e. with two different breakdown voltages. The ghost cells obtained by electrical haemolysis in the above solution containing 10mM phosphate were fairly impermeable to sucrose and behave like an ideal osometer. It is further demonstrated that ghost cells can be loaded with enzymes (e.g. urease) and drugs using this technique and that these loaded ghost cells can be used as bioactive capsules for clinical application.