Preserved erythrocyte membranes produced from slaughterhouse blood as systems for prolonged delivery of active substances

Abstract

In this doctoral dissertation, а process for production of empty erythrocyte membranes („ghosts“) originating from bovine and porcine wasted slaughterhouse blood was developed. In addition, with an aim of designing prolonged drug delivery vehicles, ghost-based drug delivery systems were formulated and in vitro characterized. In order to design the process by which hemoglobin would be released from erythrocytes, leaving preserved cell membranes, the first step was to define specific osmotic properties of bovine and porcine erythrocytes derived from slaughterhouse blood. The results showed that porcine erythrocytes were more osmotically susceptible compared to bovine ones, but neither of the two examined types of erythrocytes was homogeneous regarding their osmotic properties. This heterogeneity was detected among erythrocytes from different samples of one species, as well as among erythrocytes in one sample. The hypotonic sodiumphosphate/ NaCl buffer of 35 mM concentration circulating with a flow rate of 300 ml/h during 30 min was optimal for conducting gradual hypotonic hemolysis. Under these conditions the process of gradual hemolysis resulted with a high hemolysis extent (>90%). Biochemical analysis revealed that protein and cholesterol content of isolated ghosts were nearly the same as in intact erythrocytes. Compared to starting erythrocytes, average diameter of the ghosts from both type of erythrocytes decreased for only 10%. These results indicate that isolated membranes have possibility to mimic the chemical and structural anisotropic environment of in vivo cell membranes, and this fact is of significance for drug diffusion and partition coefficients during encapsulation procedure. In the next step, the gradual hypotonic hemolysis was evaluated as an encapsulation procedure for a model drug into the ghosts which were obtained in the previous experimental stage. A synthetic glucocorticoid, widely used antiinflammatory drug, dexamethasone sodium phosphate (DexP), was selected as a model compound. The processing parameters of DexP encapsulation into the ghosts-temperature, initial drug concentration, and cross-linker addition- were optimized...

U ovoj doktorskoj disertaciji razvijen je proces za izolovanje goveđih i svinjskih eritrocitnih membrana („duhova”) iz otpadne klanične krvi. Pored toga, razvijeni su i detaljno in vitro okarakterisani sistemi za produženo oslobađanje aktivnih supstanci na bazi dobijenih eritrocitnih membrana. U cilju projektovanja procesa kojim bi se dobile goveđe i svinjske eritrocitne membrane koje su strukturno očuvane, a poreklom iz otpadne klanične krvi, definisane su specifične osmotske karakteristike ovih eritrocita. Pokazano je da su svinjski eritroci osmotski osetljiviji i da eritrociti nijedne od ispitivanih vrsta nisu osmotski homogeni. Ova heterogenost je detektovana između eritrocita različitih uzoraka iste vrste, ali i eritrocita jednog uzorka. Kao optimalni pufer za izvođenje hemolize goveđih i svinjskih eritrocita određen je 35 mM natrijum fosfatni/NaCl pufer čijom primenom se u procesu gradualne hipotonične hemolize, pri protoku pufera od 300 ml/h, tokom 30 min, oslobađa >90% hemoglobina iz velike zapremine (100 ml) obe vrste eritrocita. Biohemijska analiza dobijenih membrana eritrocita je pokazala da se sadržaj proteina i holesterola nije bitno razlikovao u odnosu na membrane intaktnih eritrocita. Takođe, prečnik eritrocitnih membrana obe vrste bio je za samo 10% manji od intaktnih eritrocita, a raspodela veličine uniformna. Ovi rezultati su pokazali da duhovi klaničnih eritrocita obe vrste životinja imaju sposobnost da „imitiraju" hemijsko i strukturno anizotropno okruženje ćelijskih membrana in vivo, što je od značaja za difuziju i particioni koeficijet aktivnih supstanci pri njihovoj inkapsulaciji. U sledećoj fazi istraživanja ispitana je mogućnosti primene procesa gradualne hipotonične hemolize za inkapsulaciju model supstance, sintetskog glukokortikoida i najšire korišćenog antiinflamatornog leka, deksametazon-natrijum-fosfata (DexP)...