Delivery to macrophages and toxic action of etoposide carried in mouse red blood cells

Erythrocytes could be used as physiological carriers of active compounds. Several substances can be loaded into erythrocytes by hypotonic dialysis methods. Furthermore, carrier erythrocyte membrane can be chemically modified in order to promote increased arrival of the loaded compound to macrophages. In this work, we have prepared erythrocytes loaded with etoposide. We found conditions to obtain high etoposide encapsulation yields with minor alteration of some cell parameters of these carrier erythrocytes. Etoposide loaded into erythrocytes is mainly localised in the cytoplasmic compartment. Membrane modification of etoposide-loaded erythrocytes with band 3 crosslinkers produces an increased incorporation of the drug into macrophages mainly by phagocytosis process. The toxic effect of etoposide conveyed in these carrier erythrocytes determined as DNA fragmentation in macrophages was higher than that shown by free etoposide added at the same concentration in the culture medium to macrophages. These results seem to indicate the usefulness of this model to deliver this anti-tumour compound to macrophages, which might be useful in therapy.     

Intraperitoneal administration of carrier erythrocytes in dogs: an improved method for delivery of L-asparaginase

Carrier erythrocytes were prepared to encapsulate L-asparaginase by a hypotonic dialysis process. Dogs received either intravenous or intraperitoneal injections of cells containing both L-asparaginase and the marker [3H]inulin. The route of administration had no effect on the circulating survival of carrier erythrocytes. For enzyme therapy, intraperitoneal injection of a large volume of cells is technically feasible. Cells circulate with a maximum of 30% of the cells reaching circulation 24 h after injection. Those cells reaching circulation have a 7-day half-life for the encapsulated enzyme L-asparaginase.     

Delivery to Macrophages of Interleukin 3 Loaded in Mouse Erythrocytes

Mouse carrier erythrocytes containing ¹²⁵I-interleukin 3 have been prepared and treated with band 3 crosslinking reagents. The incorporation of interleukin 3 by hypotonic treatment into mouse erythrocytes reached levels of about 15% of the interleukin 3 added to the medium being predominantly present in the cytosolic fraction (73%). Uptake fell to about 7.4% when using the same conditions but omitting hypotonic shock. The interaction of band 3 crosslinked interleukin 3 loaded erythrocytes with macrophages was also studied. A high level of incorporation of interleukin 3 into macrophages was observed either from band 3 crosslinked, interleukin 3-loaded erythrocytes or from interleukin 3 loaded erythrocytes. The observations encourage the view that the system may be able to deliver and target cytokines and other growth factors to macrophages.     

Changes in allosteric properties of phosphofructokinase bound to erythrocyte membranes.

Human and rabbit erythrocyte membranes prepared by hypotonic hemolysis contained 5 to 15% of the phosphofructokinase in the erythrocytes. The membrane-bound phosphofructokinase can be eluted by a saline wash. Human erythrocyte and rabbit muscle phosphofructokinase bind to the saline-washed membranes. This binding is specific for the inner surface of the membrane. The amount of phosphofructokinase bound is dependent on pH; at pH 7, 6 times more enzyme is bound than at pH 7.5. Unlike free phosphofructokinase, the membrane-bound phosphofructokinase is not inhibited by ATP or 2,3-diphosphoglycerate, and its fructose-6-P saturation curve is nonsigmoidal.     

A disulfide-bridge bifunctional imidoester as a reversible cross-linking reagent.

A disulfide-bridged bifunctional imidoester, dimethyl 3, 3′ dithio-bispropionimidate (DTP) has been prepared and investigated as a reagent to introduce covalent cross-links in proteins that can subsequently be broken by mild reduction. Such reversible cross-links were shown to be introduced by DTP in the soluble subunit proteins aldolase and Concanavalin A. DTP was also used to modify human intact erythrocytes. Such modification rendered the erythrocytes resistant to hypotonic lysis; subsequent treatment with mercaptoethanol lysed the cells. After DTP-modification of the cells, the hemoglobin contained in them could still be reversibly oxygenated and deoxygenated.     

Encapsulation of proteins into human erythrocytes: a kinetic investigation

Moderate osmotic shocks of human erythrocytes by hypotonic dialysis (0.06 mosmol/kg) induce cell swelling and formation of pores, without causing apparent lysis. Using 125I-labeled macromolecules of different molecular weight and net charge, we followed the kinetics and efficiency of their encapsulation into erythrocytes. After a 20-30 min period of cell dialysis, macromolecules of up to 50 kDa begin diffusing into the swollen cells by a process which can be described by a first-order two-compartment kinetics. Adsorption to the external cell surface was insignificant, while adsorption to the inner membrane surface was substantial (15-20%) only for positively charged proteins, at physiological pH. After resealing, pores of a 12-14 kDa cut-off might remain open allowing some release of entrapped material (20-30%), depending on the final cytocrit, while the remaining might be associated with inner membrane or cytosolic components. Although the method of hypotonic dialysis is known to affect minimally the biophysical and immunological properties of red blood cell membranes, the interaction of encapsulated material with cell constituents would need to be further assessed when considering red cells as macromolecular carriers.     

Oral oxymetholone reduces mortality induced by gamma irradiation in mice through stimulation of hematopoietic cells

Oxymetholone is a 17α -alkylated anabolic-androgenic steroid. This drug can stimulate bone marrow cells and increase the blood cells in the peripheral blood vessels. It has been used for the treatment of anemia caused by low red cell production. Since oxymetholone has hematopoietic effect, we studied radioprotective effects of this drug in mice. In this study, we determined percentage of survival, dose-reduction factor (DRF) and hematological parameters in irradiated mice which treated with or without oxymetholone. Oxymetholone administrated at different doses 80, 160, 320, 640 mg/kg by gavages at 24 h before 8 Gy gamma irradiation. At 30 days after treatment, the following percentage of animals survival in each group was as: 80 mg/kg, 50%; 160 mg/kg, 50%; 320 mg/kg, 55%; 640 mg/kg, 75% and vehicle, 15%. Percentage of survival increased in all of treated groups statistically compared with irradiated-vehicle group. In the groups treated by oxymetholone, maximum protection was realized at 640 mg/kg. In order to calculate the DRF for oxymetholone, mice were exposed to whole-body gamma irradiation with dose ranges between 5.83 and 11.23 Gy. The probit line for oxymetholone-treated mice was shifted to the right with a DRF of 1.14. In mice exposed to whole-body gamma-irradiation (4 Gy), an oral administration of 640 mg/kg oxymetholone ameliorated radiation-induced decreases in circulating platelets and erythrocytes, but had a less effect on total number of WBC. These results demonstrate that oxymetholone stimulates myelopoiesis and thrombocytopenia and enhances survival in mice after ionizing radiation.     

Hepatic or splenic targeting of carrier erythrocytes: a murine model

Carrier mouse erythrocytes, i.e., red cells, subjected to a dialysis technique involving transient hypotonic hemolysis and isotonic resealing were treated in vitro in three different ways: (a) energy depletion by exposure for 90 min at 42 degrees C; (b) desialylation by incubation with neuroaminidase; and (c) oxidative stress by incubation with H2O2 and NaN3. Procedure (c) afforded maximal damage, as shown by analysis of biochemical properties of the treated erythrocytes. Reinfusion in mice of the variously manipulated erythrocytes following their 51Cr labeling showed extensive fragilization as indicated by rapid clearance of radioactivity from the circulation. Moreover, both the energy-depleted and the neuraminidase-treated erythrocytes showed a preferential liver uptake, reaching 50 and 75%, respectively, within 2 h. On the other hand, exposure of erythrocytes to the oxidant stress triggered a largely splenic removal, accounting for almost 40% of the reinjected cells within 4 h. Transmission electron microscopy of liver from mice receiving energy-depleted erythrocytes demonstrated remarkable erythrocyte congestion within the sinusoids, followed by hyperactivity of Kupffer cells and by subsequent thickening of the perisinusoidal Disse space. Concomitantly, levels of serum transaminase activities were moderately increased. Each of the three procedures of manipulation of carrier erythrocytes may prove applicable under conditions where selective targeting of erythrocyte-encapsulated chemicals and drugs to either the liver or the spleen has to be achieved.     

Vesiculation induced by amphiphiles in erythrocytes

The ability of shape-transforming cationic, anionic, zwitterionic, and nonionic amphiphiles to induce vesiculation in human erythrocytes was studied. At concentrations where they exhibit maximum protection against hypotonic haemolysis (CAHmax) echinocytogenic amphiphiles induced a rapid release of exovesicles. Following 5 min of incubation, the vesicle release (acetylcholinesterase release) amounted from 4% (sodium alkyl sulphates) to 13% (zwittergents) of the total acetylcholinesterase activity of the erythrocytes. At concentrations corresponding to CAH50 the vesicle release was less than 15% of that released at CAHmax. The size and the appearance of the vesicles varied with the type of amphiphile. Stomatocytogenic amphiphiles which do not pass the erythrocytes through echinocytic stages, did not induce release of exovesicles. Electron and fluorescence microscopic observations of erythrocytes treated with stomatocytogenic amphiphiles strongly indicated that an endovesiculation had occurred. Amphiphiles which pass the erythrocytes through echinocytic stages before stomatocytic shapes are attained, induced a release of both exo- and endovesicles.     

Analysis of the effects of erythrocytes on mitogen-dependent clonal proliferation of murine B lymphocytes

Addition of intact erythrocytes to semisolid agar cultures of murine B cells dramatically improves cloning efficiency and affects colony morphology. In this study, we investigated possible mechanisms through which this might occur. Specific modification of sheep erythrocyte (SRBC) membranes by treatment with trypsin but not other enzymes improved colony potentiation and erythrocytes from rats, mice, and humans were also effective after trypsin treatment. In addition, autoantibody-coated murine erythrocytes were superior to normal cells in this regard. These observations suggest that erythrocytes enhance lymphocyte survival and/or proliferation by means of particular membrane-mediated processes. The possible importance of erythrocytes as scavengers of toxic hydroxyl radicals was also investigated. Deliberately generated radicals formed by addition of dihydroxyfumaric acid and iron were effectively countered by addition of SRBC. More detailed analyses revealed that of several endogenously produced toxic species, hydrogen peroxide may be the most important under ordinary culture conditions. That is, addition of catalase but not Superoxide dismutase or mannitol improved cloning efficiency in cultures lacking SRBC. These studies suggest that erythrocytes have a beneficial effect on lymphocyte survival and function in culture through at least two mechanisms.     

A new differential technique for staining the heteropycnotic X-chromosome in female mice

The technique described here is the result of a successful attempt to identify the heteropycnotic X-chromosome at metaphase in female mice. A new modification of the commonly used air-drying technique for spreading chromosomes was employed using bone marrow cells. After treatment with hypotonic solution of 0.5% KCl at 50 °C for 30 min, all the chromosomes except one of the two X-chromosomes were only palely stained with Giemsa solution (pH 6.9) or quinacrine mustard. This X-chromosome with high stainability was not observed in males.     

Mutagenic effect of pesticides fastac 10 EK and durs ban 4E studied in a micronucleus test iin mouse bone marrow cells

The mutagenic activity of vastak and durs ban pesticides was studied by the micronucleus test in mouse bone marrow. The frequency of micronuclei in polychromatic erythrocytes was tested at 24, 36 and 42 h after oral administration of 50% LD50 dose of vastak (14 mg/kg) and durs ban (30.5 mg/kg). Significantly different increase in micronucleated polychromatic erythrocytes was established at 24, 36 and 48 h after vastak administration, and at 24 and 36 h after durs ban treatment. Doses of 25% LD50 for both pesticides showed no mutagenic activity, as judged by the induction of micronuclei in polychromatic erythrocytes.     

Inhibition by nucleosides of glucose-transport activity in human erythrocytes.

The interaction of nucleosides with the glucose carrier of human erythrocytes was examined by studying the effect of nucleosides on reversible cytochalasin B-binding activity and glucose transport. Adenosine, inosine and thymidine were more potent inhibitors of cytochalasin B binding to human erythrocyte membranes than was D-glucose [IC50 (concentration causing 50% inhibition) values of 10, 24, 28 and 38 mM respectively]. Moreover, low concentrations of thymidine and adenosine inhibited D-glucose-sensitive cytochalasin B binding in an apparently competitive manner. Thymidine, a nucleoside not metabolized by human erythrocytes, inhibited glucose influx by intact cells with an IC50 value of 9 mM when preincubated with the erythrocytes. In contrast, thymidine was an order of magnitude less potent as an inhibitor of glucose influx when added simultaneously with the radioactive glucose. Consistent with this finding was the demonstration that glucose influx by inside-out vesicles prepared from human erythrocytes was more susceptible to thymidine inhibition than glucose influx by right-side-out vesicles. These data, together with previous suggestions that cytochalasin B binds to the glucose carrier at the inner face of the membrane, indicate that nucleosides are capable of inhibiting glucose-transport activity by interacting at the cytoplasmic surface of the glucose transporter. Nucleosides may also exhibit a low-affinity interaction at the extracellular face of the glucose transporter.     

The analysis of recessive lethal mutations in mice by using two-dimensional gel electrophoresis of liver proteins

We have used two-dimensional gel electrophoresis (2DE) coupled with computer-assisted data analysis to analyze liver-protein expression in mice known to be heterozygous carriers of recessive lethal mutations induced in In(1)1Rk or In(7)13Rk inversion stocks by exposure to either triethylene melamine or ionizing radiation. Carriers of 8 different mutations and corresponding littermate controls (average of 17 individuals in each group) were screened for liver-protein differences. Both qualitative and quantitative protein differences were detected that correlated with unique pedigrees among the mouse stocks analyzed. Such strain-specific differences demonstrated that quantitative differences (either increases or decreases) in protein abundance of greater than 25% can be readily detected by using this 2DE system. Thus the 50% reduction in expression of a protein expected in the event of a structural gene deletion is well within the level of detection. No significant quantitative decreases in protein expression that correlated with the recessive lethal mutations were detected, however.     

Influence of mouse age and erythrocyte age on glutathione metabolism.

In order to determine whether the biological age of a mouse influences erythrocyte metabolism and erythrocyte aging in vivo, blood samples were collected from male C57/BL6J mice of different biological ages ranging from mature (10 months) to "very old" (37 months). In the very old mouse, compared with the mature mouse, the erythrocyte survival time was decreased, erythrocyte densities were increased, the concentrations of total free thiol and reduced glutathione, and glutathione reductase activity were decreased. Erythrocytes were separated into different density (age) groups by phthalate ester two-phase centrifugation or by albumin density-gradient centrifugation. The density-age relationship of erythrocytes was established by pulse-labelling with 59Fe in vivo and by subsequent determinations of specific radioactivity of erythrocyte fractions of different densities prepared during a chase period of 60 days. The age of erythrocytes in mice of all ages was directly related to density. Also, in older erythrocytes compared with younger erythrocytes, decreased concentrations of total free thiol and reduced glutathione, and decreased glutathione reductase activity were observed. These were the lowest in the old erythrocytes of very old mice. These results in aging erythrocytes from aging mice suggest that the glutathione status the erythrocyte may be an index of aging, not only of the cell but also of the organism.     

Membrane fluidity change in erythrocytes induced by complement system.

The structural change in erythrocyte membranes induced by antibody and complement was studied using phospholipid spin-labels. Sheep erythrocytes were labeled with phosphatidylcholine spin-label and various intermediate cells (erythrocyte-antibody complex (EA), EA bound with complement components from C1 to C7 (EAC1-7), EAC1-8, and EAC1-9) were prepared. Electron spin resonance spectra of EA, EAC1-7, and EAC1-8 were very similar to that of the erythrocytes, while that of EAC1-9 was markedly different. The overall splitting value for the lysed EAC1-9 (53 G) was much smaller than that for the erythrocytes (57 G), indicating a marked fluidization around the phosphatidylcholine label. The unlysed EAC1-9 membranes contained a limited fraction of the fluidized area. When EA was reacted with complement in the presence of 36% bovine serum albumin, the membranes were fluidized similarly to the lysed EAC1-9, although the hemolysis was largely blocked. The membranes of unlysed EAC1-9 prepared in isotonic (ethylenedinitrilo)tetraacetic acid were also fluidized, but to somewhat smaller extent. The role of C9 in the modification of erythrocyte membranes was also demonstrated using Mg2+ ghosts, which were prepared by hypotonic hemolysis in the presence of Mg2+. The membranes of Mg2+ ghost of EAC1-7 were markedly fluidized when bound with C8 and C9, but not affected by binding of C8 only. The component C8 was found to give a latent effect on the membranes that caused irreversible fluidization upon osmotic shock. The terminal component thus creates a fluidized area in the erythrocyte membranes through which small ions and molecules may diffuse more easily and the resulting osmotic unbalance may finally cause hemolysis.     

Deformability of stored erythrocytes as a criteria for their in vivo survival rate

The loss of deformability observed in erythrocytes stored as whole blood for 36 days (ACD-AG) or as buffy-coat free erythrocyte concentrate (EK) was characterized by measuring their filterability. During the first 3 weeks the index of filterability for ACD-AG erythrocytes increased only slightly and rose to about 140% of its initial value on the 36th day. In contrast, a heavy loss of deformability (increase of the filterability index to more than 600%) was detected for erythrocytes from EK, which, from a rheological point of view, is apt to raise doubts of using this stored blood. An incubation of 1 hour at 37 degrees C in fresh plasma did not result in improving the deformability. A cell volume loss of more than 20% connected with an increase of the inner viscosity to more than 400% was found to be the cause of this decrease of deformability. These rheological differences are also reflected in the 24 hours in vivo survival rate (SR), if the "early loss" of damaged erythrocytes immediately after transfusion is taken into account. Whereas the SR values of 80% for whole blood erythrocytes do not change significantly, the SR values for EK values can be found to reach 54% approximately.     

Inhibition and stimulation of K+ transport across the frog erythrocyte membrane by furosemide, DIOA, DIDS and quinine

Frog erythrocytes were incubated in iso- or hypotonic media containing 10 mmol/l Rb+ and 0.1 mmol/l ouabain and both Rb+ uptake and K+ loss were measured simultaneously. Rb+ uptake by frog red cells in iso- and hypotonic media was reduced by 30-60% in the presence of 0.01-0.1 mmol/l [(dihydroindenyl)oxy] alkanoic acid (DIOA) or 0.5-1.0 mmol/l furosemide. Furosemide inhibited K+ loss from frog erythrocytes incubated in hypotonic media but did not affect it in isotonic media. DIOA at a concentration of 0.05 mmol/l inhibited of K+ loss from frog erythrocytes in both iso- and hypotonic media. At the concentrations of 0.01 and 0.02 mmol/l DIOA significantly suppressed K+ loss in a K+-free chloride medium but not in a K+-free nitrate medium. The Cl(-)-dependent K+ loss was completely blocked at a concentration of 0.1 mmol/l DIOA and the concentration required for 50% inhibition of K-Cl cotransport was approximately 0.015 mmol/l. However, the inhibitory effect of DIOA on K-Cl cotransport was masked by an opposite stimulatory effect on K+ transport which was also observed in nitrate medium. Quinine in a concentration of 0.2-1.0 mmol/l was able to inhibit Rb+ uptake and K+ loss only in hypotonic media. In isotonic media, quinine produced a stimulation of Rb+ uptake and K+ loss. A three to five-fold activation of Rb+ uptake and K+ loss was consistently observed in frog erythrocytes treated with 0.05-0.2 mmol/l 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). In contrast, another stilbene derivative 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) had no effect on K+ transport in the cells. Thus, of these drugs tested in the present study only DIOA at low concentrations may be considered as a selective blocker of the K-Cl cotransporter in the frog red blood cells.     

Ultrastructural observations on the transformations of osmotically stressed and electrically pulsed red cell carriers at stages during their formation

Although annealed red blood cell (RBC) carriers, when made by different methods, encapsulate similar quantities of methotrexate (MTX) molecules, the mechanisms by which the carrier cells are formed and by which molecules are taken up may be very different as evidenced by electron microscopy (EM) of the cells at various stages during their formation. Scanning electron microscopy (SEM) revealed that dialysis- and preswell-prepared carriers, both procedures which involve exposure to hypotonic buffer, initially transform from discocytes to echinocytes, but later exhibit morphologically heterogeneous cell types. In contrast, electroporated carriers, formed under isotonic conditions, uniformly show an initial discocyte-spheroechinocyte transformation. Transmission electron microscopy (TEM) of osmotically stressed RBC carriers demonstrates that uptake of molecules can be facilitated both by endocytosis and passive diffusion, whereas electrically pulsed carriers encapsulate material only by passive diffusion. Such experiments provide evidence that methods can be refined potentially for producing and isolating carriers that would have more predictable properties in vivo.     

The Erythrocyte Ghost Is a Perfect Osmometer

The osmotic swelling of intact erythrocytes in hypotonic solutions was measured using microhematocrit tubes, Van Allen tubes, and a calibrated Coulter counter. In agreement with earlier workers the intact cells did not behave as perfect osmometers, the cells swelling less than predicted by the Boyle-van't Hoff law. Erythrocyte ghosts were prepared from fresh intact erythrocytes by one-step hemolysis in 0.25% NaCl at an extremely dilute concentration of cells and the membranes were sealed at 37°. The ghosts were mixed with NaCl solutions of different osmolarities and the MCV (mean cell volume) of the shrunken cells immediately monitored by a calibrated Coulter counter. It was found that the MCV values of the shrunken ghosts were accurately predicted by the Boyle-van't Hoff law. These results indicate that these erythrocyte ghosts behaved as perfect osmometers.