José Luque

Erythrocytes as Carriers for Recombinant Human Erythropoietin

AbstractPurpose. The aim of this work was to encapsulate recombinant human erythropoietin (rHuEpo) in human and mouse red blood cells (RBCs) to improve the stability of encapsulated rHuEpo. Methods. The encapsulation of rHuEpo was achieved by an hypotonic dialysis-isotonic resealing procedure. A radioimmunoassay method was used for the estimation of rHuEpo. The hypoosmotic resistance of carrier erytrhocytes was studied by osmotic fragility measurements. Cell morphology was observed under scanning electron microscopy. Encapsulated rHuEpo was identified by an immunogold labeling assay. Results. Encapsulation yields were 22% for human RBCs and 14% for mouse RBCs. Cell recovery was around 70%. Carrier-RBCs exhibited a tendency to spherocytic morphology, and showed the typical higher hypoosmotic resistance than normal RBCs. The presence of rHuEpo inside carrier RBCs was identified. The stability of encapsulated rHuEpo seems to be related to the experimental conditions used during the encapsulation procedure. An increase with time of released rHuEpo was observed in carrier-RBC suspensions. Conclusions. The encapsulation of rHuEpo in RBCs has been achieved for the first time. These carrier RBC-preparations may serve as an alternative sustained cell delivery system for the in vivo administration of rHuEpo.

In vitro and in vivo study of glutamate dehydrogenase encapsulated into mouse erythrocytes by a hypotonic dialysis procedure

Glutamate dehydrogenase (GDH) has been encapsulated into mouse erythrocytes by a hypotonic dialysis/isotonic resealing method. Although a low GDH entrapment yield was achieved (3.8%), this percentage appeared sufficient enough to metabolize high quantities of ammonia. Carrier cell recovery yield was 56%. Due to the decrease in cell volume and haemoglobin content, constant mean cell haemoglobin concentration (MCHC) values were obtained. The osmotic fragility curves (OFC) indicated that dialyzed/resealed-RBCs are more resistant to hypotonic haemolysis than native-RBCs. The successful in vitro ammonia degradation by GDH-RBCs was reflected in its total disappearance from the incubation medium at around 48 h. In contrast, initial ammonia levels were not affected during the incubation in the presence of native-RBCs and remained constant. Two different methods were used for the preparation of hyperammonaemic mice model. Since the intraperitoneal (i.p.) administration of ammonium acetate produced high ammonia levels that lasted only a few minutes, the i.p. administration of urease was chosen, given that it generated elevated ammonia levels for longer periods of time. Hyperammonaemic mice quickly removed high levels of circulating ammonia in the presence of GDH-RBCs, whereas in the presence of native-RBCs ammonia was slowly metabolized. These results suggest that loaded GDH-erythrocytes can be used as a potential carrier systems for the in vivo removal of high levels of ammonia from blood.

IN VITRO STUDY OF ALCOHOL DEHYDROGENASE AND ACETALDEHYDE DEHYDROGENASE ENCAPSULATED INTO HUMAN ERYTHROCYTES BY AN ELECTROPORATION PROCEDURE

The optimal conditions for electroporated/resealed loading of alcohol dehydrogenase (ADH) and/or acetaldehyde dehydrogenase (ALDH) into human erythrocytes were established prior to the study, with the following characteristics: 300 V, 1 ms pulse time, eight pulses every 15 min and 1 h resealing at 37 degreesC. High encapsulation yield and carrier cell recoveries were achieved. Cell volumes increase while hemoglobin contents decrease; in consequence a decrease in cell hemoglobin concentration was observed. A lower hypotonic resistance of loading erythrocytes (throughout osmotic fragility curves) and unaltered oxygen transport capability (as given by oxygen equilibrium curves) were observed. The stability against time (up to 168 h-7 days) of encapsulated individual enzymes, either ADH- or ALDH-red blood cells (RBCs), was studied at 4 degreesC and 37 degreesC, in comparison with that of free enzyme solutions. Both enzymes were released from carrier RBCs to the incubation medium. The stability of carrier RBCs was studied under similar conditions. Non-significant variations in hematological parameters were observed. However, the hemoglobin derivative forms showed modifications. The continuous degradation of ethanol by ADH-RBCs and coencapsulated ADH- and ALDH-RBCs, as a function of time (up to 70 h) suggests the use of these carrier RBCs as agents for complete metabolization of ethanol. The mentioned properties bare the possibility of using ADH and ALDH as carrier systems in in vivo situations.

Partial purification of glucose 6-phosphate dehydrogenase and phosphofructokinase from rat erythrocyte haemolysate by partitioning in aqueous two-phase systems.

Glucose 6-phosphate dehydrogenase shows a high partition coefficient in poly-(ethylene glycol)-dextran aqueous two-phase systems in comparison with those for 6-phosphogluconate dehydrogenase, phosphofructokinase and the bulk of proteins present in rat erythrocyte haemolysates. As a consequence, fractions highly enriched in glucose 6-phosphate dehydrogenase can be obtained after multiple partitions in the above systems with a counter-current distribution procedure. Phosphofructokinase shows a high affinity for Cibacron Blue and, as a result, the enzyme can be extracted in the top phase of poly(ethylene glycol)-dextran systems containing Cibacron Blue-poly(ethylene glycol) (affinity systems). The efficiency for the purification of the enzymes by partitioning is increased up to 10-fold when enzyme-rich fractions, obtained by precipitation with poly(ethylene glycol), are used instead of original haemolysate. The recovery of enzyme activities is near 100% in both instances.

Bisphosphoglycerate mutase and pyruvate kinase activities during maturation of reticulocytes and ageing of erythrocytes

An increase in bisphosphoglycerate mutase (BPGM) and a decrease in pyruvate kinase (PK), i.e. a decrease in PK/BPGM ratio, was observed in red cell populations from anemic rats containing 95% down to 3% reticulocytes in blood. Such a ratio has been used to study the fractionation of recticulocytes, according to their degree of maturation, after counter-current distribution of those cell populations in dextrahpoly (ethylene glycol) two-phase systems. When applying this procedure to the fractionation according to age of erythrocytes from normal rats, the decrease of PK with cellular age was observed without a significant variation in BPGM activity.

Ligand-receptor interactions in affinity cell partitioning: Studies with transferrin covalently linked to monomethoxypoly(ethylene glycol) and rat reticulocytes

The partitioning of rat reticulocytes in poly(ethylene glycol) (PEG)-dextran two-phase systems increases into the PEG-rich top phase when the cells are incubated with transferrin covalently modified with monomethoxy-PEG (MPEG-transferrin) prior to partitioning. Two observations support the suggestion that such an increase in top-phase partitioning is due to the specific interaction of the MPEG-transferrin conjugate with the transferrin receptor on the surface of the reticulocyte: first, the MPEG-transferrin conjugate competes with [125I]transferrin for the transferrin receptor on reticulocytes (Ka = 6.28 x 10(6) l mol-1); and second, the MPEG-modified transferrin is unable to change the partitioning of rat erythrocytes, cells lacking the transferrin receptor. This example illustrates the feasibility of manipulating the partitioning of a selected cell population when ligand-receptor interactions are exploited. The increase in the partitioning of the reticulocytes takes place within a narrow range of MPEG-transferrin bound per cell, viz., 10.2-11.3 fg per cell. The latter range corresponds to ca. 80,000-89,000 molecules of MPEG-transferrin bound per cell.

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.

Analysis by partitioning in aqueous two-phase systems of the loss of transferrin-binding capacity during maturation of rat reticulocytes

A decrease in the number of binding sites for125I-transferrin, without an apparent modification of the association constant, has been observed during the maturation of reticulocytes into erythrocytes. As an experimental model, different red cell populations from phenylhydrazinic anaemic rates (95% to 12% reticulocyte-rich) have been used. The fractionation by multiple partition in two-phase systems of these red cell populations has been applied here to show the relationship between number of transferrin receptors and rate of reticulocyte maturation.

Settling-time dependence of rat bone marrow cell partition and counter-current distribution in charge-sensitive aqueous two-phase systems : Relationship with the cell partitioning mechanism

Differences in the settling-time dependence of single and multiple cell partitions have been found between heterogeneous (bone marrow cells) and homogeneous (erythrocytes) populations when using charge-sensitive dextran-poly(ethylene glycol) aqueous two-phase systems. The cell populations were partitioned using both single test-tube experiments and multiple thin-layer counter-current distribution. Lengthening the settling time, to favour phase separation, and decreasing the upper phase volume are more effective in fractionation by the counter-current distribution of heterogeneous cell populations than increasing the interfacial tension, although all three were employed to speed phase settling. On the basis of these results, the original cell partitioning mechanism proposed for non-charge-sensitive systems has been extended to charge-sensitive systems.

Affinity partitioning of erythrocytic phosphofructokinase in aqueous two-phase systems containing poly(ethylene glycol)-bound Cibacron Blue influence of pH, ionic strength and substrates/effectors

Phosphofructokinase (PFK) from rat erythrocyte haemolysates has a high affinity for Cibacron Blue F3G-A covalently bound to poly(ethylene glycol) (PEG-Cb) and thus the enzyme can be extracted into the top phase of poly(ethylene glycol)-dextran aqueous two-phase systems containing PEG-Cb. The pH, ionic strength and presence of substrates/effectors affect to different extents the affinity of the enzyme for PEG-Cb and the number of PEG-Cb molecules attached per molecule of PFK (the latter probably reflecting, at saturation, the influence on the aggregation state of the enzyme) and thus influence the yield of enzyme recovered in the top phase. Increasing the pH from 6 to 7 and then to 8 leads to a higher yield of PFK in the top phase. A change in pH from 6 to 7 and 8 results in an increased number of PEG-Cb molecules attached per molecule of enzyme while the affinity of PFK for PEG-Cb shows a minimum at pH 7. The ionic strength in the range 0.017-0.164 has less influence on the partitioning of PFK. The presence of substrates or effectors of the enzyme in general reduces the recovery of PFK in the top phase. Fructose 6-phosphate increases the number of PEG-Cb molecules attached but greatly reduces the affinity of PFK for PEG-Cb. In contrast, AMP slightly reduces the number of PEG-Cb molecules attached and the affinity of PFK for PEG-Cb. ATP and ATP-Mg2+ compete with PEG-Cb for the same binding sites in PFK.(ABSTRACT TRUNCATED AT 250 WORDS)