JoséC. Díez

In vitro properties and organ uptake of rat band 3 cross-linked erythrocytes

Chemical conditions for cross-linking rat erythrocytes with bis(sulfosuccinimidyl)suberate (BS3) and 3,3 dithiobis(sulfosuccinimidyl propionate) (DTSSP) have been studied. These two cross-linking reagents seem to react with band 3 proteins in rat erythrocyte membrane. Two different conditions have been assayed using different cell/cross-linker concentration ratios. Similar cell volumes were observed in cross-linked rat erythrocytes compared to rat control erythrocytes. Cell yields after cross-linking account for about 65% when a high ratio of cell-to-cross-linker was used. Slightly lower cell yields (about 62%) were obtained when this ratio was reduced. Estimation of band 3 cross-linking by gel electrophoresis revealed a level of cross-linking of around 45% and 50% at the different conditions used. In vivo behavior of these modified rat erythrocytes revealed that they do not circulate, showing a predominant localization in the liver. This effect is evident from the concentration (5 mM BS3 or DTSSP) used. Based on these results, BS3 and DTSSP can be considered as useful tools to cross-link rat erythrocyte band 3 and to target rat erythrocytes to the liver.

TARGETING OF MOUSE ERYTHROCYTES BY BAND 3 CROSSLINKERS

Chemical conditions of crosslinking mouse erythrocytes with BS3 and DTSSP have been studied. These two crosslinking reagents seem to react with band 3 protein in mouse erythrocytes membrane. Extent of crosslinking is dependent on the concentration of the reagent used. Similar cell volumes were observed in crosslinked erythrocytes with respect to control erythrocytes. In vivo behaviour of these modified erythrocytes revealed prominent targeting of crosslinked erythrocytes to liver. This effect is clearly evident when concentrations of 5 mM BS3 or DTSSP were used and can be dependent of reagent concentration. Consequently, from our results BS3 and DTSSP can be considered as very useful tools to control and modulate targeting of crosslinked erythrocytes.

IN VIVO BEHAVIOUR OF RAT BAND 3 CROSS-LINKED CARRIER ERYTHROCYTES

Rat band 3 cross-linked carrier erythrocytes have been prepared. Iodinated carbonic anhydrase has been encapsulated into rat erythrocytes. Then, carrier erythrocytes were labeled with 51chromium. Eventually, these doubly labeled rat RBCs were treated with a band 3 cross-linking reagent, namely bis(sulfosuccinimidyl)suberate (BS3). 51Chromium labeling and 125I CA showed to have cytosolic localization in cross-linked carrier erythrocytes. Estimation of the band 3 cross-linking induced by BS3 on rat carrier erythrocytes has been done rendering values around 25% of band 3 monomer reduction. BS3-cross-linked carrier erythrocytes when injected into rats are mainly targeted to liver as shown by chromium labeling localization. Also, encapsulated CA radioactivity carried by cross-linked carrier rat erythrocytes when injected into rats is localized predominantly in liver as shown by in vivo experiments. Accordingly, cross-linked carrier erythrocytes are highly recognized by peritoneal macrophages as detected by in vitro analyses of macrophage recognition. Thus, our data revealed a targeting of carrier rat erythrocytes induced by cross-linking of band 3 protein by BS3. These results support claims in favor of this animal model as a feasible system to analyze cross-linked carrier erythrocytes survival and targeting as well as the in vivo efficacy of targeting of loaded compounds to liver.

Differential association of the different brain microtubule proteins in different in vitro assembly conditions

Microtubule protein was assembled in vitro for different time periods and at different protein concentrations near and far from the critical concentration needed for assembly. When the isolated polymers were characterized by electrophoresis, a higher association of high-molecular-weight microtubule-associated proteins, particularly of the larger microtubule-associated protein, was found in the polymers assembled close to the critical concentration, while tau factor polypeptides are predominantly present in those polymers assembled at higher protein concentration. Also, a higher proportion of high-molecular-weight microtubule associated proteins MAP1 and MAP2 was observed in microtubules assembled for short time periods, compared with those obtained after the monomer-polymer equilibrium was reached. When the assembled protein was further characterized by isoelectric focusing, no differences were found in the proportion of the different isotubulins present in the polymers assembled under different conditions tested.

Rat erythrocyte glycophorins can be isolated by the lithium diiodosalicylate method used for other glycophorins

1. The lithium diiodosalicylate/phenol method, widely employed for the isolation of membrane sialoglycoproteins (glycophorins) from mammalian erythrocytes, was applied for the first time to the purification of homologous glycoproteins from rat erythrocyte membranes. 2. The resulting preparations showed to be composed of four components, fractionated on SDS-PAGE. All four were positive for periodic acid-Schiffs reagent stain, the two largest of them being major. 3. Isolated rat glycophorins accounted for 60% of the ghost sialic acid and 1.5% of their protein. The presence of O-acetyl groups was confirmed in one-third of the sialic acid residues. 4. The molecular masses of the four glycophorin components were determined by a method which takes into account the anomalous mobility of glycoproteins on SDS-electrophoresis. Estimated values thus obtained for the actual molecular masses were 74, 32, 25 and 17 kDa.

Influence of Chemical Modification on “ In Vivo ” and “ In Vitro ” Mouse Carrier Erythrocyte Survival and Recognition

Several systems have been developed with therapeutical purposes for drug delivery. Among them, red blood cells (RBCs) have been claimed to be a physiological method to convey and deliver active compounds.4,6,8,11 The preparation of erythrocytes as carriers can require encapsulation procedures or/and chemical modification of erythrocyte surface.3,22 In fact, the efficacy of these systems can be dependent of the use of several chemical treatments which can react with cell membrane proteins. Crosslinking reagents can be applied to red blood cell modification. Glutaraldehyde (GA) has been the most extensively used crosslinker.7,21 Also, other crosslinkers can be applied to carrier erythrocytes preparation.12,16 Biotinylation is another alternative method for carrier preparation.15,19 Eventually, chemical modification can promote targeting of carrier erythrocytes to several organs.12,21 We have focused our attention on the action of crosslinking reagents which react with band 3 in mouse erythrocyte membrane. Additionally, biotinylation of mouse erythrocytes has been studied. “In vivo” behaviour of these chemically modified erythrocytes have been studied. These survival results have been compared to recognition by macrophages. Thus, we described conditions for using these chemical treatments for targeting to organs and macrophages.

Immunoanalysis and quantitation of membrane sialoglycoproteins (glycophorins) in rat erythrocytes and reticulocytes.

An enzyme-linked immunosorbent assay (ELISA) for the quantitation of rat erythrocyte membrane sialoglycoproteins (glycophorins) has been developed. Samples of erythrocytes and reticulocytes were analysed using this assay, and response compared among them and purified glycophorins samples. A broadly homologous behaviour of glycophorins was found in both cell types, suggesting the presence in reticulocytes of glycophorin molecules closely similar to those on the erythrocyte. A quantitative evaluation of glycophorins on both cell types yielded comparable levels of these glycoproteins, but with significantly higher values (1.7-fold) for reticulocytes. It is suggested that the lower number of epitopes present on the erythrocyte membrane might be due to the disappearance of some glycophorin-associated antigenic determinants during the maturation of reticulocyte to erythrocyte.

Immunological relationship among rat erythrocyte membrane sialoglycoproteins and with human glycophorins

1. Specific antibodies against a whole preparation of glycophorins from rat erythrocyte membrane and against its most prominent component (32 kDa) were prepared. 2. Both antisera and the respective affinity-purified antibodies recognize the 74, 32 and 25 kDa components of rat glycophorins; therefore, a close antigenic relationship between them can be inferred. 3. On the other hand, a cross-reaction with human glycophorin A (both MM- and NN-type) is observed for both antisera. 4. This suggests the existence of epitopes common to human and rat glycophorins.

Rat Carrier Erythrocytes Circulate and Arrive to Organs

Different delivery systems are currently used in therapy. They have the advantage of protecting the active substance from rapid clearance and avoiding toxic side effects. Among the many carrier systems proposed12, RBCs have many desirable properties: they are naturally biodegradable and may stay in circulation over prolonged periods of time11,25; RBCs are easily obtainable and large amounts of material can be entrapped in a small volume of cells by hypotonic dialysis; autologous cells elicit little or no immune response7,16,20.