Hypotonic Hemolysis Research Articles

Biopharmaceutical characteristics of autologous red blood cells ghosts containing cytokines and antibiotics.

IntroductionTransport systems based on autologous red blood cells for targeted drug delivery can be considered as a promising approach in the treatment of surgical infections. Experimental studies have revealed the feasibility of targeted drug delivery by encapsulation of cytokines and antibiotics into autologous erythrocyte ghosts.PurposeTo study biopharmaceutical characteristics of autologous erythrocyte ghosts containing cytokines and antibiotics (pharmacocytes).Material and methodsThe erythrocyte pharmacocytes were prepared by the hypotonic hemolysis method, or the use of human red blood cells. The association and dissociation indicators of rifampicin and cytokine substances with the erythrocyte ghosts were conducted using standard methods.ResultsWe have defined the following extracellular concentrations to be optimal for deposition of drug substances into pharmacocytes: for rifampicin – 10 000 μ/ml, erythropoietin - 1000 IU/ml, TNF-a - 5000 IU/ml, IL-1-β - 5000 U/ml, IFN-γ - 10 000ME/ml, IL-2 - 50 000 IU/ml, angiogenin - 0.04 mg/ml. Two types of correlations of cytokines and pharmacocytes were identified. In this study, we found that 40–60 % of the erythropoietin, IFN- γ and angiogenin were bound to red blood cells ghosts, more than 10% of which were bound irreversibly. For TNF-a, IL-1-β and IL-2, the red blood cells ghosts were capable of binding and depositing within 10–20 % of the input extracellular concentration, and these bindings were almost completely reversible. The rifampicin was bound by red blood cells ghosts with 5 % efficiency and also completely reversibly.ConclusionThe study has shown the effectiveness of inclusion of the studied components, such as erythropoietin, IFN-γ and angiogenin into the red blood cells ghosts, with significant efficiency (40–60 %). It presents the potential of using this system in targeted delivery of cytokines and antibiotics for treatment of surgical infections, thus facilitating the reduction in toxicity and adverse systemic effects of drugs and improving the treatment results.

Enrichment isolation of adipose-derived stem/stromal cells from the liquid portion of liposuction aspirates with the use of an adherent column

Background aimsAdipose-derived stem/progenitor cells (ASCs) are typically obtained from the lipoaspirates; however, a smaller number of ASCs can be isolated without enzymatic digestion from the infranatant liposuction aspirate fluid (LAF). We evaluated the effectiveness of an adherent column, currently used to isolate mesenchymal stromal cells from bone marrow, to isolate LAF cells. MethodsWe applied peripheral blood (PB), PB mixed with cultured ASCs (PB-ASC), and LAF solution to the column and divided it into two fractions, the adherent (positive) and the non-adherent (negative) fractions. We compared this method with hypotonic hemolysis (lysis) for the red blood cell count, nucleated cells count and cell compositions as well as functional properties of isolated mesenchymal cells. ResultsThe column effectively removed red blood cells, though the removal efficiency was slightly inferior to hemolysis. After column processing of PB-ASC, 60.5% of ASCs (53.2% by lysis) were selectively collected in the positive fraction, and the negative fraction contained almost no ASCs. After processing of LAF solution, nucleated cell yields were comparable between the column and hemolysis; however, subsequent adherent culture indicated that a higher average ASC yield was obtained from the column-positive samples than from the lysis samples, suggesting that the column method may be superior to hemolysis for obtaining viable ASCs. Mesenchymal differentiation and network formation assays showed no statistical differences in ASC functions between the lysis and column-positive samples. ConclusionsOur results suggest that a column with non-woven rayon and polyethylene fabrics is useful for isolating stromal vascular fraction cells from LAF solutions for clinical applications.

The release of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from human erythrocyte membranes lysed by hemolysin of Prevotella oris

We found that a 38-kDa protein was released from erythrocyte membranes lysed by hemolysin of Prevotella oris, although hypotonic hemolysis did not show such a phenomenon. The 38-kDa protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by N-terminal amino acid sequencing. This study discusses the relationship between GAPDH and hemolysis.

Synthesis of new chiral xanthone derivatives acting as nerve conduction blockers in the rat sciatic nerve

The synthesis and structure elucidation of three new chiral xanthone (9H-xanthon-9-one) derivatives (2–4) are fully reported. The coupling reactions of the synthesized building block 6-methoxy-9-oxo-9H-xanthene-2-carboxylic acid (1) with two enantiomerically pure amino alcohols ((S)-(+)-valinol and (S)-(+)-leucinol) and one amine ((S)-(−)-α-4-dimethylbenzylamine), were carried out using the coupling reagent O-(benzotriazol-1-yl-)-N,N,N′,N′-tetramethylluronium tetrafluoroborate (TBTU). The coupling reactions were performed with yields higher than 97% and enantiomeric excess higher than 99%. The structures of the compounds were established by IR, MS, and NMR (1H, 13C, HSQC, and HMBC) techniques. Taking into account that these new chiral xanthone derivatives have molecular moieties structurally very similar to local anaesthetics, the ability to block compound action potentials (CAP) at the isolated rat sciatic nerve was also investigated. Nerve conduction blockade might result from a selective interference with Na+ ionic currents or from a non-selective modification of membrane stabilizing properties. Thus, the mechanism, by which the three chiral xanthone derivatives cause conduction blockade in the rat sciatic nerve and their ability to prevent hypotonic haemolysis, given that erythrocytes are non-excitable cells devoid of voltage-gated Na+ channels, are also described. Data suggest that nerve conduction blockade caused by newly-synthesized xanthone derivatives might result predominantly from an action on Na+ ionic currents. This effect can be dissociated from their ability to stabilize cell membranes, which became apparent only upon increasing the concentration of compounds 2–4 to the higher micromolar range.

Dielectric spectroscopy reveals nanoholes in erythrocyte ghosts

When blood is diluted with water, erythrocytes swell and then burst to release haemoglobin molecules, viz. hypotonic hemolysis. The remaining membranes, so-called “ghosts”, have transient holes, which are resealed under physiological conditions. About 50 years ago it was reported that ghost suspensions showed peculiar dielectric dispersion below 10 kHz, termed α-dispersion, which was not found for intact erythrocyte suspensions. The finding, however, has never been traced because of difficulty in low-frequency measurement due to electrode polarization (EP) effects, and therefore the origin of the α-dispersion has not been understood. In this study, the α-dispersion has been revealed using a new type of measurement cell capable of reducing the EP effects. The properties of the α-dispersion were exactly interpreted by modelling ghosts as a spherical cell with a single hole. The numerical simulation with the cell model provided a linear relation between the characteristic frequency of the α-dispersion and the hole radius, the hole radius thereby being determined straightforwardly.

Surface-modified loaded human red blood cells for targeting and delivery of drugs

Red blood cells (RBCs) are natural carriers which can be used for targeted drug delivery. Conditions during loading and surface modification are essential for carrier-RBC preparation for specifically targeted drug delivery. Therefore, human RBCs were loaded with albumin and magnetic nanoparticles (NPs) by different hypotonic haemolysis procedures and compared based on loading efficiency and membrane damage. Samples were analysed by flow cytometry and confocal microscopy. The optimized loading procedure resulted in 90% albumin-loaded carrier-RBCs with <4% Annexin V binding and 263 pg iron per RBC after loading with iron oxide NPs. Albumin-loaded RBCs were subsequently surface conjugated with insulin and IgG via biotin–streptavidin. Insulin-conjugated carrier-RBCs were observed to attach and to be internalized by cultured endothelial cells. Uptake was not observed for carrier-RBCs non-specifically modified with IgG. Attachment of other peptides with high specificity will open novel opportunities for targeting various cells, tissues and for crossing biological barriers.

Investigations on Membrane Perturbation by Chrysin and Its Copper Complex Using Self-Assembled Lipid Bilayers

The mechanism of membrane interactions of most of the flavonoids in the presence of transition-metal ions is not well-understood. To understand this phenomenon, the present work aims to synthesize a chrysin-copper complex at room temperature and investigate its influence on the electrical characteristics of planar lipid bilayers. The chrysin-copper complex was characterized by various spectroscopic techniques and was found to have a metal/ligand ratio of 1:2 and of cationic nature. Its ability to inhibit 1,1'-diphenyl-2-picrylhydrazyl (DPPH) radicals was not significant at alkaline pH because of the involvement of the 5-hydroxy group in coordination with the copper ion compared to its parent flavonoid, chrysin (p < 0.05). The addition of different concentrations (20-100 μM) of chrysin and the chrysin-copper complex to lipid bilayers decreases the resistance, indicating a strong surface interaction and partial insertion into the bilayer near the lipid-water interface. The dose-dependent reduction in resistance as a result of the chrysin-copper complex is more pronounced in comparison to chrysin, implying that the bulkier and charged chrysin-copper complex displays greater ability to distort the lipid bilayer architecture. These conclusions were further confirmed by curcumin-loaded liposome permeabilization studies, where both chrysin and its Cu(II) complex increased the fluidity in a dose-dependent manner. However, the extent of fluidization by the chrysin-copper complex was nearly twice that of chrysin alone (p < 0.05). The implications of these surface interactions of chrysin and its copper complex on cell membranes were studied using a hypotonic hemolysis assay. Our results demonstrate that, at low concentrations (20 μM), the chrysin-copper complex exhibited twice the protection against hypotonic stress-induced membrane disruption when compared to chrysin. However, this stabilizing effect gradually decreased and became comparable to chrysin at higher concentrations. This biphasic behavior of the chrysin-copper complex could further be explored for therapeutic applications.

In vitro Inhibition of Fungal Activity by Macrophage‐Mediated Sequestration and Release of Encapsulated Amphotericin B Nanosupension in Red Blood Cells

The efficacy of antifungal treatment has been diminished by the biodistribution limitations of amphotericin B (AmB) due to its pharmacological profile, as well as the severe side effects it causes. A cellular drug-delivery system, which incorporates human erythrocytes (RBCs) loaded with an AmB nanosuspension (AmB-NS), is developed in order to improve antifungal treatment. AmB-NS encapsulation in RBCs is achieved by using hypotonic hemolysis, leading to intracellular AmB amounts of 3.81 +/- 0.47 pg RBC(-1) and an entrapment efficacy of 15-18%. Upon phagocytosis of AmB-NS-RBCs, leukocytes show a slow AmB release over ten days, and no alteration in cell viability. This results in an immediate, permanent inhibition of intra- and extracellular fungal activity. AmB-NS-RBC-leukocyte-mediated delivery of AmB is efficient in amounts 1000 times lower than the toxic dose. This drug-delivery method is effective for the transport of water-insoluble substances, such as AmB, and this warrants consideration for further testing.

The haemolytic effect of verapamil on erythrocytes exposed to varying osmolarity

The haemolytic effect of verapamil on red blood cells (RBCs) exposed to varying osmolarity was investigated. The experimental approach used a modified red cell haemolysis assay with concentrations of verapamil ranging from 50–1500 μM compared to drug free controls. The time-course of haemolytic effects was also investigated. We also briefly determined the haemolytic effects of verapamil in Ca 2+-free conditions (with added EGTA). In conditions representing decreasing osmolarity (dilution from 140–0 mM NaCl) there was a significant increase in erythrocyte haemolysis that was also dependent on verapamil concentration (ANOVA, p < 0.05). The red cells also showed a significantly increased rate of haemolysis over 5 h with increasing verapamil concentration (ANOVA, p < 0.05). The degree of RBC hypotonic haemolysis was significantly increased in a Ca 2+-free medium (+EGTA) compared to normal saline and this effect was exacerbated by additions of verapamil (ANOVA, p < 0.05). Overall the data suggested that verapamil can cause haemolysis of RBCs in a predictable time- and concentration-dependent manner, and that verapamil increases the fragility of the erythrocytes further during hypotonic osmotic stress and Ca 2+-free conditions. The mechanism of verapamil-dependent haemolysis could be directly related to the observed biphasic concentration-effect and could consequently involve several ion transport pathways.

Hemolysis and antihemolysis induced by amino acid-based surfactants

Surfactants have the special ability to interact with the lipid bilayer of cell membranes. The red blood cell is one of the most used cellular membrane models to study the mechanisms underlying surfactant-induced osmotic cell resistance. To increase our knowledge regarding the mechanisms of surfactant membrane interaction, we studied the action of five lysine-derivative anionic and three arginine-derivative cationic amino acid-based surfactants on hypotonic hemolysis. Results showed two different antihemolytic behaviors among amino acid-based surfactants, both related to the maximal protective concentration. How the physico-chemical properties and structure of these compounds determine the protection against hypotonic hemolysis is discussed in detail. We found a good correlation between the CMC and the concentrations resulting in maximum protection against hypotonic hemolysis for the cationic surfactants, but no correlation for the anionic surfactants. In the case of lysine derivative surfactants, which only differ in their counterions, the counterion is implicated in the differences in the antihemolytic potency and the hemolytic activities of this.

In vivo effect of aluminium upon the physical properties of the erythrocyte membrane

Aluminium (Al (III)) is a metal with no biological function. Its organic accumulation can lead to toxic effects. To elucidate the in vivo effect of Al (III) upon the rheological properties of the erythrocyte membrane, male adult Wistar rats have been submitted to periodical injections of Al(OH) 3 during three months. Significant decreases in haematocrit (34 ± 0.37% versus 36 ± 0.20%, p < 0.0001) and blood haemoglobin concentration (10.7 ± 0.15 g/dl versus 12.3 ± 0.49 g/dl, p < 0.005) have been found. Haemolysis curves shifted towards the left, indicating that erythrocytes became more resistant to hypotonic haemolysis. Significant increments in rigidity index (29.6 ± 1.59 versus 9.2 ± 0.40, p < 0.0001), relative viscosity at native haematocrit (3.6 ± 0.03 versus 3.5 ± 0.03, p < 0.04), and relative viscosity at standard haematocrit (4.5 ± 0.06 versus 3.9 ± 0.05, p < 0.0001) have been observed. The decrease in the erythrocyte aggregate size (1.6 ± 0.01 versus 1.7 ± 0.01, p < 0.002) and the aggregation rate (0.5 ± 0.02 versus 0.6 ± 0.03, p < 0.002) indicated a significantly dropped aggregability process. In conclusion, Al (III) disorganised the erythrocyte membrane by altering its mechanical properties, suggesting a reduction of the middle life of circulating erythrocytes, which could play a major role in the anaemia of these animals.

Interaction of electrically neutral and cationic forms of imipramine with liposome and erythrocyte membranes

Imipramine (IP) is an amphiphilic amine that is clinically useful as a tricyclic antidepressant. In the present work, we have investigated the pH effects on (a) the micellization properties of IP, (b) IP-induced release of aqueous contents from artificial vesicles (liposomes), and (c) IP-induced haemolysis, under hypotonic and isotonic conditions, in the pH range 5.0–10.0. Our data show that IP-membrane interaction caused important membrane destabilisation. However, changes in pH modified strongly the effects of IP. We suggest that the parameters of IP-induced model and cell membrane lysis are sensitive to the changes in the charge of this amphiphile. IP interacts more strongly with the membrane at basic pH (smaller net charge) than at neutral or acidic pH. A simple interpretation of these data is that increasing pH causes a decrease in IP net charge, thus a decrease in the critical micellar concentration of IP, and an increased partition into the lipid bilayer. Concomitantly, pH variations both above and below 7 tend to destabilise the cell membrane, so that the protective effect of IP against hypotonic haemolysis has a maximum at neutral pH.

Erythrocyte membrane permeability for a series of diols

Erythrocyte membrane permeability coefficients for a series of diols have been defined by the method developed. The method is based on the physical and mathematical modeling of hypotonic hemolysis process. There have been also determined membrane permeability coefficients for erythrocytes treated with p-chloromercuribenzenesulfonic acid monosodium salt (pCMBS), which is known to block aqueous protein channels. Permeating process is shown to be conditioned both by hydrophilic/hydrophobic properties of the molecules and their geometrical parameters. The obtained results propose that, when exceeding the molecules diameter over a value of 4 Å, the permeability coefficient reduces due to decreasing of flow through the aqueous protein pores of a constant size. Permeability coefficients for comparatively hydrophobic molecules are almost directly proportional to the coefficients of partition between hydrophobic and hydrophilic phases, by pointing to a lipid way of permeation of these molecules through erythrocyte membranes.

The study of the quercetin action on human erythrocyte membranes

Quercetin is a naturally occurring flavonoid that exerts multiple pharmacological effects. In our previous study, we showed that quercetin greatly affects the lipid membrane. In this report, a study of quercetin on human erythrocyte membrane has been performed to determine the influence of this flavonoid on the fluidity and the conformational changes of membrane proteins. An additional aim of the study was to find how quercetin presence affects the resistance of membrane to haemolytic agents. The results showed that incorporation of quercetin into the erythrocyte membranes caused the changes of the partition coefficient of the Tempo spin label between the water and polar head group phases. In the studies, the W/S ratio has been used as a monitor of changes in protein conformation and in the environment within the membrane. It was observed that quercetin caused an increase in protein–protein interactions in human erythrocyte membranes. Haemolytic action of quercetin in the dark was also investigated. This compound showed protective effect against hypotonic haemolysis. However, in the heat-induced haemolysis quercetin caused acceleration of haemolysis. Dark reaction of erythrocyte with quercetin resulted in a shrinkage of the cells and alteration of their shapes. From the results we have concluded that modification of erythrocyte membrane by quercetin proceeds via reaction with membrane lipids and proteins.

Effects of the Antiplatelet Agent TA-993 and Its Metabolite MB3 on the Hemorheological Properties of Rat and Human Erythrocytes

In the present study, we investigated the effects of the antiplatelet agent TA-993 and its metabolite MB3 on the hemorheological properties of rat and human erythrocytes in comparison with ticlopidine and aspirin. TA-993 and MB3 concentration-dependently lowered the viscosity of rat erythrocyte suspensions. TA-993 and MB3 inhibited both the hypotonic hemolysis of human erythrocytes and the mechanical hemolysis of rat erythrocytes induced by turbulent flow. Treatment of rats with TA-993 (10 mg/kg/day po) for 10 days significantly increased blood filterability, but ticlopidine and aspirin did not show this effect. TA-993 and MB3 enhanced the interaction of 1-anilino-8-naphthalene sulfonate (ANS), a hydrophobic probe, with human erythrocyte ghosts and reduced the fluorescence polarization in 1,6-diphenyl 1,3,5-hexatriene (DPH, a fluidity probe)-labeled human erythrocyte ghosts. TA-993 and MB3 induced aggregation of liposome suspensions prepared from acidic phospholipids. These findings suggest that TA-993 and MB3 may affect the erythrocyte membrane by interacting with acidic phospholipids and thus improve the hemorheological properties.

Effect of Chlorpromazine on hypotonic and isotonic hemolysis

Effect of Chlorpromazine on hypotonic and isotonic hemolysis

Gemini (dimeric) surfactant perturbation of the human erythrocyte.

We studied the ability of di-cationic gemini surfactantsdi (amphiphiles), i.e. 1,4-butanediammonium-N,N-dialkyl-N,N,N',N'-tetramethyl bromides (Di-Cm-di-QAS (s = 4), where m = 8, 11, 13, 16 and s = the number of alkyl groups in the spacer) to induce shape alteration, vesiculation, haemolysis and phosphatidylserine exposure in human erythrocytes, and to protect erythrocytes against hypotonic haemolysis. At high sublytic concentrations the Di-Cm-di-QAS (s = 4) amphiphiles rapidly induced echinocytic (spiculated) shapes and a release of exovesicles, mainly in the form of tubes, from the cell surface. Following 60 min incubation erythrocytes were sphero-echinocytic and a few cells with invaginations/endovesicles were observed. No phosphatidylserine exposure was detected. The haemolytic potency increased with an increase of the alkyl chain length. At sublytic concentrations the Di-Cm-di-QAS (s = 4) amphiphiles protected erythrocytes against hypotonic haemolysis. It is suggested that the Di-Cm-di-QAS (s = 4) amphiphiles perturb the membrane in a similar way as single-chain cationic amphiphiles, but that they do not easily translocate to the inner membrane leaflet.

Inhibition of glucose transport and direct interactions with type 1 facilitative glucose transporter (GLUT-1) by etomidate, ketamine, and propofol: A comparison with barbiturates

Ketamine, etomidate, propofol, and pentobarbital were compared for effects on and interactions with the type 1 facilitative glucose transporter (GLUT-1). Fluxes of radiolabeled hexoses were used to determine the effects of anesthetics on GLUT-1 function. Hypotonic hemolysis of human erythrocytes was used to assess perturbations of membrane integrity. Quenching of intrinsic protein fluorescence was used to assess the direct interactions of anesthetics with purified GLUT-1. Pentobarbital, ketamine, etomidate, and propofol inhibited glucose transport in murine fibroblasts with ic 50 values of 0.8, 1.6, 0.1, and 0.4 mM, respectively. Pentobarbital, ketamine, etomidate, and propofol also inhibited sugar transport in human erythrocytes. The ic 50 values for pentobarbital and ketamine exhibited substrate dependence for equilibrium exchange but not unidirectional effluxes. This was not observed for etomidate. Propofol did not inhibit equilibrium exchange but did inhibit unidirectional efflux with little substrate dependence. Pentobarbital protected against hemolysis, whereas etomidate and ketamine promoted hemolysis of erythrocytes. Propofol had no effect on membrane integrity. Pentobarbital, ketamine, and etomidate all interacted directly with GLUT-1, with apparent K d values of 2.2, 0.8, and 0.5 mM, respectively. Like barbiturates, ketamine, etomidate, and propofol inhibited GLUT-1 at concentrations near to those used pharmacologically. Inhibition of GLUT-1 by these intravenous general anesthetics was complex, exhibiting differential kinetic effects on equilibrium exchange versus unidirectional fluxes and contrasting substrate dependencies. Like barbiturates, ketamine and etomidate bound to GLUT-1 with affinities that paralleled inhibition of glucose transport. As a class, intravenous general anesthetics, in contrast to inhalation anesthetics, inhibit GLUT-1-mediated glucose transport.

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.

Erythrocyte hemolysis and shape changes induced by new lysine-derivate surfactants

The effects of new synthetic lysine-derived anionic surfactants on human and rat erythrocytes were studied. The surfactants were salts of N α, N ε-dioctanoyl lysine with different counterions: lysine (77KK), tris (trishydroxymethyl amminomethane) (77KT), sodium (77KS), and lithium (77KL). 77KK and 77KT showed a biphasic hemolytic behavior in the erythrocytes. The surfactants 77KS and 77KL showed concentration-dependent hemolysis with a CH50 of about 3.4 and 2.6 mmol/l, respectively. 77KK and 77KT induced protection against hypotonic hemolysis in rat erythrocytes at the concentration which showed the least hemolytic activity under isotonic conditions. With human erythrocytes, 77KT did not show biphasic behavior in isotonic medium, but under hypotonic conditions biphasic behavior was present. Changes in shape of the erythrocyte, from discocytic to stomatocytic were observed after incubation with the anionic surfactants studied. Such shape changes occurred progressively over time, with total alteration in shape occuring after about 20 min of incubation.