Ivana Kostić

Flow cytometric determination of osmotic behaviour of animal erythrocytes toward their engineering for drug delivery

Despite the fact that the methods based on the osmotic properties of the cells are the most widely used for loading of drugs in human and animal erythrocytes, data related to the osmotic properties of erythrocytes derived from animal blood are scarce. This work was performed with an aim to investigate the possibility of use the flow cytometry as a tool for determination the osmotic behaviour of porcine and bovine erythrocytes, and thus facilitate the engineering of erythrocytes from animal blood to be drug carriers. The method of flow cytometry successfully provided the information about bovine and porcine erythrocyte osmotic fragility, and made the initial steps in assessment of erythrocyte shape in a large number of erythrocytes. Although this method is not able to confirm the swelling of pig erythrocytes, it indicated to the differences in pig erythrocytes that had basic hematological parameters inside and outside the reference values. In order to apply/use the porcine and bovine erythrocytes as drug carriers, the method of flow cytometry, confirming the presence of osmotically different fractions of red blood cells, indicated that various amounts of the encapsulated drug in porcine and bovine erythrocytes can be expected.

Interaction of cobalt(II), nickel(II) and zinc(II) with humic-like ligands studied by ESI-MS and ion-exchange method

The interactions of Co(II), Ni(II) and Zn(II) with humic acid and O-donor humic-like ligands were studied by the ion-exchange and electrospray ionization mass spectrometry (ESI-MS) methods. Interactions were confirmed by differences between the values of chromatogram peak areas for monocom- ponent (ligand) and binary systems (ligand with metal ion) by a newly deve- loped ESI-MS 5 µL-loop injection technique. The Schubert ion exchange method was used for the determination of the stability constants of the formed complexes at pH 4.0 and I = 0.01 mol dm -3 . Comparing the values of log K for complexes formed by di-positive metal ions with humic, benzoic and salicylic acids, it could be concluded that the interaction strengths of d-metals followed the Irving-Williams order: Co(II) Zn(II). The obtained values of log K indicated that Pb(II) ions affect the strongest interactions with all the investigated ligands. Complexation of humic acids macromolecules possessing O-donor binding sites as major binding sites could be predicted and modeled following the same order of interaction strength as the Irving-Wil- liams order.

Optimization of gradual hemolysis for isolation of hemoglobin from bovine erythrocytes

In this work, we describe an optimized procedure based on gradual hemolysis for the isolation of hemoglobin derived from bovine slaughterhouse erythrocytes in a membrane bioreactor. The membrane bioreactor system provided high yields of hemoglobin (mainly oxyhemoglobin derivate) and its separation from the empty erythrocyte membranes (ghosts). Ten different concentrations of hypotonic media were assessed from the aspect of the extent of hemolysis, hematocrit values of the erythrocyte suspensions, cell swelling and membrane deformations induced by decreased salt concentration. Effective gradual osmotic hemolysis with an extent of hemolysis of 83% was performed using 35 mM Na-phosphate/NaCl buffer of pH 7.2-7.4. Under these conditions most of the cell membranes presented the appearance of the normal ghosts under phase contrast microscope. The results show that isolation process yielded predominantly to oxyhemoglobin. Kinetic studies showed that maximal concentration of hemoglobin was reached after 40 min, but the process cycle at which recovery of 83% was achieved lasted for 90 min.