Carrier erythrocytes from cattle can be prepared from cells stored in a hypotonic, porous, dialyzed state

Hypotonic dialysis of bovine erythrocytes to 160-180 mosmol/kg produces cells with metastable pores. Cells stored for 3 weeks in a hypotonic dialyzed state can be used to prepare carrier erythrocytes. Pores generated by nonlytic hypotonic conditions are initially greater than 40 A in radius but shrink to less than 20 A with storage beyond 1 week. A metastable pore allows for hemoglobin isolation from hypotonically dialyzed cells.     

A single partitioning step in aqueous polymer two-phase systems reduces hypotonized rat erythrocyte heterogeneity

Rat carrier erythrocytes prepared by hypotonic dialysis (80 mOsm/kg) are a heterogeneous cell population that can be fractionated into two-well-defined cell subpopulations by a single partition step, in charge-sensitive dextran-poly(ethylene glycol) aqueous two-phase systems. One subpopulation (65% of total cells) has a decreased cell surface charge and is partitioned at the interface in a single step and then fractionated by counter-current distribution as a low-G subpopulation. The other subpopulation (35% of total cells) has charge surface properties more like those of the untreated control rat erythrocytes. These last cells are partitioned in the top phase in a single step and then fractionated by counter-current distribution as a high-G subpopulation. Partitioning is more effective in reducing cell heterogeneity in hypotonized rat erythrocyte populations than is density separation in Ficoll-paque which only separates a small less dense cell subpopulation (5% of total cells), with the most fragile cells, from a larger and more dense cell subpopulation (95% of total cells), with a mixture of fragile and normal cells. This simple cell separation procedure quickly reduces carrier erythrocyte heterogeneity in a single partitioning step so it can be used to prepare cells for in vivo studies.     

Determination of parameters for enzyme therapy using L-asparaginase entrapped in canine erythrocytes

The antitumor agent L-asparaginase was entrapped in canine erythrocytes by a single dialysis encapsulation (efficiency mean = 30%). Concentration of asparaginase in carrier cells was about 240 IU/ml, with an average of 62% cell recovery. Use of a double dialysis procedure increased the L-asparaginase concentration within carrier cells to 530 IU/ml, with an overall cell recovery of 53.9%. In vitro efflux experiments showed L-asparaginase-loaded canine carriers were stable at both 4 and 37 degrees C for an 18-h period. In vivo cell survival studies showed that carrier cells did circulate and that L-asparaginase had a half-life of 6.5 days. No evidence suggesting that the enzyme left the cell was found. Carrier cells prepared with [3H]inulin and [14C]sucrose were stored at 4 degrees C for 2 weeks and began to show signs of deterioration after 2 days.     

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.     

Membrane pores in hypotonically dialyzed sheep erythrocytes remain open after three weeks of storage: entrapment of different stokes radius probes, [14C]sucrose and [3H]inulin

Sheep carrier erythrocytes were prepared from dialyzed cells stored for 3 weeks. The initial pore size in freshly dialyzed cells exceeds the Stokes radius of that for hemoglobin. Hypotonically dialyzed erythrocytes are then very stable in a porous state. Two probes of different Stokes radius were used to determine the relative size of the pores. Sheep erythrocytes entrap inulin to a greater extent than sucrose, a much smaller molecule. With storage, a greater fraction of dialyzed cells become impermeable to inulin than to sucrose indicative of pore size greater than 5.2 less than 20 A. Since hemoglobin content did not change relative to storage, the pore size was less than the Stokes radius of hemoglobin. Pores generated by controlled hypotonic dialysis are unlike the single rupture pore found in erythrocyte ghosts.     

Determination of the content of nonfilterable cells in erythrocyte suspensions as a function of the medium osmolality

The results of most filtration assays for deformability of erythrocytes do not distinguish whether the entire population or only its small fraction exhibits abnormal rheological properties. We developed a simple filtration method for determination of the percentage of nonfilterable cells in erythrocyte suspension using membrane filters with mean pore diameter of 3.1 microns. This method makes it possible to detect even minor abnormal subpopulations in erythrocyte suspensions. The flow rate of buffer depends on the number of free pores of a filter. The plot of the number of pores clogged by nonfilterable cells vs the total number of erythrocytes that were allowed to pass through the filter had a linear portion, with a slope representing the relative content, Z%, of nonfilterable cells in the suspension. We determined Z% for various medium osmolalities u and used the data to derive the distribution of erythrocytes in ucr (ucr is the maximum value of u at which an erythrocyte cannot pass through a pore of a given filter because of geometric limitations). The distribution of ucr in suspension of normal erythrocytes has a maximum of about 200 mOsm/kg and a half-width of about 20 mOsm/kg. The distributions of ucr are altered in normal erythrocyte suspensions at decreased pH values, in cryopreserved and ATP-depleted erythrocyte suspensions and in erythrocytes from a xerocytosis patient.     

大鼠红细胞作为SOD新型载体的细胞水平上的研究

用低渗透析 -等渗重封的方法制备了包埋超氧化物歧化酶 ( SOD)的大鼠载体红细胞 ,并从细胞水平上研究了透析条件对大鼠红细胞包埋 SOD的影响与载体红细胞的部分性质 .流式细胞计( FCM)研究表明 ,随透析时间延长和透析液渗透压降低 ,包埋 SOD的载体红细胞百分率升高 ,但载体细胞平均包埋 SOD的量无明显变化 ;SOD浓度对载体细胞百分率无明显影响 ,但与载体细胞平均包埋 SOD的量成线性关系 ;载体红细胞前向角散射 ( FLS)明显下降 ,但显微镜下观察到的载体细胞的大小无明显变化 ,当载体细胞反注射到大鼠体内后 FLS能迅速恢复 ;载体红细胞密度下降 ,其原因是低渗透析时红细胞膨胀未能完全恢复 ;载体红细胞未暴露与自身 Ig G结合的抗原位点 .激光扫描共聚焦显微镜 ( LSCM)分析表明 ,SOD在细胞内呈从细胞中心到细胞膜浓度逐步下降的辐射分布特征 .     

Encapsulation by hypotonic dialysis in human erythrocytes: a diffusion or endocytosis process

Human erythrocytes subjected to controlled hypotonic dialysis are capable of encapsulating and retaining drugs. Under selected conditions encapsulation has been reported to occur by an endocytosis process. The mechanism by which encapsulation occurs under conditions which are conducive for endocytosis to occur was studied. An analysis of the percentage of cells with endocytic vacuoles was made for cells dialyzed to optimal and suboptimal osmotic pressures for encapsulation. No differences were found with approximately 20% of cells from all preparations containing vacuoles. Transmission electron micrographs of cells in different stages of carrier cell preparation reveal endocytic vacuoles both with and without hemoglobin. However, based on the percentage of exogenous substance encapsulated, encapsulation appears to occur primarily by diffusion and secondarily by endocytosis.     

Plasmodium berghei: Osmotic fragility of malaria parasites and mouse host erythrocytes

The osmotic properties of intraerythrocytic and ultrasonically liberated malaria parasites (Plasmodium berghei) were analyzed and compared with those of mouse host erythrocytes utilizing a multiple tube fragility test. Cells were incubated in phosphate buffered saline solutions of varying osmolalities ranging from 20–4000 mOsm. Changes in cell ultrastructure and parasite infectivity were used as indicators of osmotic damage. Intraerythrocytic and host cell-free plasmodia showed similar patterns of cell alteration and changes in infectivity following osmotic stress. The various developmental forms within each of the preparations responded somewhat differently to hypo-osmotic stress, however. The majority of merozoites seemed to be more sensitive than many trophozoites, schizonts, and segmenters. Small trophozoites were, on the average, more resistant than other developmental forms. Incubation of parasite populations in hypotonic salt solutions with osmolalities slightly greater than the infectivity threshold of 100 mOsm lysed the majority of the merozoites, whereas many small trophozoites were still intact. While normal erythrocytes were more resistant to hypo-osmotic stress than were either intracellular or free parasites, the majority of parasitized erythrocytes was less resistant than normal erythrocytes. The predominant alteration induced by hyperosmotic stress appears in the parasite's nuclear region with myelination of the nuclear membranes and chromatin clumping. The infectivity threshold in the hypertonic range was found to be approximately 2500 mOsm. Results indicate that these obligate intracellular parasites have a wide range of osmotic sensitivities and that they are capable of existing for short periods in various osmotic environments ranging from 100–2500 mOsm without complete loss of infectivity. This suggests that these parasites have osmotic regulatory capabilities at least comparable to those of host cells.     

Volume-sensitive K transport in human erythrocytes

Studies have been carried out on human erythrocytes to examine the alterations of K transport induced by swelling or shrinking the cells by osmotic and isosmotic methods. Hypotonic swelling of erythrocytes (relative cell volume, 1.20) resulted in a striking, four- to fivefold augmentation in the ouabain-resistant K influx over the value obtained at a normal cell volume. Shrinking the cells in hypertonic media resulted in a small but statistically significant reduction in K influx. Three different methods of varying cell volume gave similar results. These include the addition of sucrose and of NaCl to hypotonic media and the isosmotic (nystatin) method. The major fraction of the K influx in swollen cells is specific in its requirement for Cl or Br and is not supported by thiocyanate, iodide, nitrate, methylsulfate, or acetate. Bumetanide (0.1 mM), MK-196 (0.2 mM), and piretanide (1 mM) are poorly effective in suppressing K uptake in swollen cells, but at higher concentrations, bumetanide (1 mM) inhibits 80% of the Cl-dependent K influx in swollen cells. The bumetanide concentration required to inhibit 50% of the Cl-dependent K influx is 0.17 mM. The volume-sensitive K influx is independent of both extracellular and intracellular Na, so that the (Na + K + 2Cl) cotransport pathway is not a likely mediator of the volume-sensitive K transport. A variety of inhibitors of the Ca-activated K channel are ineffective in suppressing swelling-induced K influx. Like K uptake, the efflux of K is also enhanced by cell swelling. Swelling-activated K efflux is Cl dependent, is independent of extracellular and intracellular Na, and is observed with both hypotonic and isosmotic methods of cell swelling. The activation of K efflux by cell swelling is observed in K-free media, which suggests that the volume-sensitive K transport pathway is capable of net K efflux. The addition of external K to hypotonic media resulted in an increase in K efflux compared with the efflux in K-free media, and this increase was probably due to K/K exchange. Thus, hypotonic or isosmotic swelling of human erythrocytes results in the activation of a ouabain-resistant, Cl-dependent, Na-independent transport pathway that is capable of mediating both net K efflux and K/K exchange.     

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.     

Elevated polyamine levels in erythrocytes of SLC:ICR mice inoculated with ehrlich ascites carcinoma cells

Ehrlich ascites carcinoma cells (4×10⁵ cells/mouse) were inoculated intraperitoneally in 7-week-old SLC:ICR mice, and polyamine levels in peripheral erythrocytes and in ascites cells were determined periodically. Polyamine levels in peripheral erythrocytes increased linearly until 10 days after cell inoculation, while ascites cells showed exponential growth.The effect of carbazilquinone on cellular growth and polyamine levels in erythrocytes was also studied. When 1 or 2mg/kg of carbazilquinone was injected intraperitoneally on day 4 or on day 7, cellular growth was suppressed and the survival time of the mice was lengthened. The polyamine levels in erythrocytes were also markedly decreased 3 days after the carbazilquinone injection.These results suggest that the polyamine levels in peripheral erythrocytes are closely related to the cellular growth of Ehrlich ascites carcinoma cells.     

Cold shock hemolysis in human erythrocytes studied by spin probe method and freeze-fracture electron microscopy.

When human erythrocytes are osmotically stressed or chemically treated, they hemolyze on cooling below 10 degrees C (called cold shock). We have studied the effects of osmotic stress and cooling on the state of membrane by the spin-probe method and freeze-fracture electron microscopy. At room temperature, the membrane fluidity detected by 12-doxyl stearate spin probe showed a steady decrease with osmolality in hypertonic NaCl solutions up to 900 mOsm/kg, above which it remained unchanged. In hypertonic sucrose solutions, the electron paramagnetic resonance spectra showed an additional pair of absorptions, indicating development of regions, in the membrane, further immobilized than in NaCl solutions. Mobility of a cholesterol analogue probe, androstane, did not show change by hypertonicity, but the spectral intensity dropped at 1,200 mOsm/kg, probably due to formation of loose aggregates in the cholesterol phase. On cooling the osmotically stressed cells in NaCl solution, the isotropic rotational correlation time vs. inverse temperature plot of 12-doxyl stearate probe exhibited a step-wise discontinuity at approximately 10 degrees C, suggestive of a drastic transition in the state of the membrane. At about the same temperature, the freeze-fracture pattern of osmotically stressed cells revealed the development of large wrinkles and aggregation of membrane particles, in contrast to the case of the cells in isotonicity. Significance of these findings in understanding cold shock hemolysis is discussed.     

A modification of the filtration method for studying the content of nonfilterable cells in erythrocyte suspension

The results of filtration assays provide estimates of the deformability of erythrocytes averaged over the entire suspension. These assays do not distinguish whether the entire population or only its small fraction exhibits abnormal rheological properties. We developed a simple method using a filtrometer to determine the percentage of non-filterable (under given conditions) cells in the erythrocyte suspension. Membrane filters made of a polyethylene terphthalate film had the mean pore diameter of 3.1 microns and the length of cylindrical micropores of 7 microns. The buffer flow rate tb depends on the number of free pores in a filter. The plot of the number of pores clogged by non-filterable cells versus the total number of erythrocytes passed through the filter had a linear portion whose slope represents the relative content Z of non-filterable cells in the suspension. We determined Z for various medium osmolarities u. These data were used to derive the distribution of erythrocytes in ucr, the value of u at which an erythrocyte cannot pass through a pore of a given filter because of geometric limitations. The distribution maximum corresponded to 190-200 mOsm/kg for erythrocytes from the normal blood. This means that normal erythrocytes have the median values of their surface area and area-to-volume ratio of 155-151 microns2 and 1.72-1.68 microns-1, respectively. The half-width of the distribution was approximately 30 mOsm/kg. This finding suggests that the normal blood contains a certain fraction of erythrocytes with a decreased area-to-volume ratio. Our results showed that the distribution is altered in various forms of anemia and in ATP-depleted erythrocyte suspensions.     

Some effects of the trypanocidal drug isometamidium on encapsulation in bovine carrier erythrocytes

Bovine erythrocyte exposure to isometamidium chloride causes increased osmotic fragility. Control cells tolerated up to 1 mg/ml drug with no effects. Carrier erythrocytes were highly susceptible to drug, with increased osmotic fragility and decreased encapsulation potential of sucrose and inulin. Scanning electron micrographs of control and carrier erythrocytes exposed to drug revealed the formation of enkephalocytes with carrier erythrocytes. Control erythrocytes showed greater tolerance to the drug. Apparently, access of the drug to the interior of the erythrocyte membrane allows the drug to be more interactive with the membrane.     

A continuous-flow high-yield process for preparation of lipid-free hemoglobin

Hypotonic hollow-fiber dialysis of bovine red blood cells followed by ultrafiltration through 0.1-micron pore hollow fibers provides a simple method for isolation of lipid-free hemoglobin. Hemoglobin (Hb) isolated by comparative techniques were all contaminated with membrane stroma. HPLC analysis of Hb revealed a protein peak of 99.6% purity and sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis revealed a single band. The process requires hypoosmotic dialysis of bovine RBC to a final 160-180 mosmol/kg osmotic pressure. Additional reduction in osmotic pressure causes irreversible cell lysis which leads to lipid contamination of the Hb. Processing of 1/2 liter of packed red blood cells requires 4-5 h, resulting in an average of 90% hemoglobin recovery.     

Actin cytoskeletons regulate the stretch-induced increase of Ca2+ current in human gastric myocytes

Using the whole-cell and single channel recording techniques, the influence of actin cytoskeletons on L-type Ca2+ current was investigated in human gastric smooth muscle cells. In isotonic condition, an actin depolymerizer cytochalasin D (Cyt-D) markedly decreased the whole-cell current (I(Ba)) without changing steady-state voltage dependency and single channel conductance. Intracellular dialysis of phalloidin, an actin polymerizer, significantly increased the I(Ba). Hypotonic stretch (222 mOsm/L) of the myocytes increased the I(Ba), and Cyt-D significantly inhibited the I(Ba) increase by the stretch. Phalloidin was without effect on the I(Ba) increase by the stretch. Phalloidin antagonized the Cyt-D inhibition of the stretch-induced I(Ba) increase. Neither heterotrimeric G protein modifiers (GTPgammaS and GDPbetaS) nor rho GTPase inhibitor (C3 exoenzyme) influenced the stretch-induced responses. These results reveal that the integrity of the actin cytoskeleton is an important factor which determines the activity of L-type Ca2+ channels and a response to stretch.     

Effect of hypoosmotic stress on hybridoma cell growth and antibody production

To investigate the response of hybridoma cells to hypoosmotic stress, S3H5/gamma2bA2 and DB9G8 hybridomas were cultivated in the hypoosmolar medium [Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% serum] resulting from sodium chloride subtraction. Both hybridomas showed similar responses to hypoosmotic stress in regard to cell growth and antibody production. The cell growth and antibody production at 276 mOsm/kg were comparable to those at 329 mOsm/kg (standard DMEM). Both cells grew well at 219 mOsm/kg, though their growth and antibody production were slightly decreased. When the osmolality was further decreased to 168 mOsm/kg, the cell growth did not occur. When subjected to hyperosmotic stress, both cells displayed significantly enhanced specific antibody productivity (q(Ab)). However, the cells subjected to hypoosmotic stress did not display enhanced q(Ab). Taken together, both hyperosmotic and hypoosmotic stresses depressed the growth of S3H5/gamma2bA2 and DB9G8 hybridomas. However, their response to hypoosmotic stress in regard to q(Ab) was different from that to hyperosmotic stress. (c) 1997 John Wiley & Sons, Inc. Biotechnol Biong 55: 565-570, 1997.     

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.     

Factors affecting the hemolytic action of "lysolecithin" upon rabbit erythrocytes

1. Lysolipid was prepared by the action of snake venom on egg yolk, and a study was made of the factors affecting its hemolytic action upon rabbit erythrocytes. 2. Lysis proceeded very rapidly at first, then ceased within a few minutes at room temperature. A given amount of lysin appeared to hemolyze a fixed number of cells, under specified conditions. 3. The more dilute erythrocyte suspensions required relatively more lysin per cell, for 50 per cent hemolysis of the suspension. There may be an equilibrium between the lysin dissolved in the medium and that adsorbed on the cells. 4. The degree of hemolysis for varying lysin concentrations was measured, and the cells showed a typical distribution of resistance to hemolysis. 5. As the temperature was lowered lysis was more extensive. Adsorption of the lysin on the cell surface was apparently increased. 6. The resistance of the erythrocytes to lysis increased slightly as the pH was raised from 5.5 to 7.8. 7. Resistance to lysis was independent of the tonicity of the medium and of initial cell volume. The magnitude of the cell surface was probably the determining factor. 8. A marked shrinkage of the erythrocytes was observed in the presence of calcium ions and lysin, but not in the absence of the lysin. 9. Hemolytic resistance curves obtained by the Wilbrandt technique were of the "colloid-osmotic" type. However, there was no evidence of prolytic loss of potassium ions. 10. Hypotonic fragility of the cells was slightly increased in the presence of the lysin. The rate of penetration of thiourea was greatly increased.