Hypo-osmotic Dialysis Research Articles

Red Blood Cells are Appropriate Carrier for Coagulation Factor VIII.

Aims Factor VIII (FVIII) replacement therapy remains a primary treatment for hemophilia A, however, the development of FVIII antibodies (inhibitors) and short half-life of the FVIII products are the major complications. Erythrocytes may prevent rapid removal of drugs from plasma. Erythrocytes are biocompatible and non-immunogenic drug delivery. In this study, in vitro activity of FVIII encapsulated by human erythrocytes was investigated.Methods FVIII was loaded into erythrocytes using the hypo-osmotic dialysis technique. FVIII activity assay has been analyzed using Activated Partial Thromboplastin Time (APTT). Presence of FVIII on erythrocytes was detected by western blotting and flowcytometry using specific monoclonal antibody (abcam, U.K) against FVIII. Moreover, the osmotic fragility and hematologic parameters of FVIII-loaded carrier erythrocytes were measured.Results Our results indicated that FVIII could not cross the membrane, where plenty of FVIII was found on the surface of the carrier erythrocyte. Flow cytometery results showed that 11% of the loaded carrier erythrocytes was positive for FVIII protein on their surface.The greatest activation of FVIII in both groups including lysate and non-lysate FVIII-loaded RBCs was observed on the first day, and the coagulant activity of this factor was gradually reduced on days 3 and 5. In 1:50 dilution of both groups, significant differences in FVIII activity were observed in 1:50 dilution of both groups, especially on the 5th day.Conclusion This study aims to introduce erythrocytes as appropriate carriers for FVIII to prolong the dosing intervals in the effective and safe levels for a relatively longer time.

Ways to Increase the Activity of Glutamate Dehydrogenase in Erythrocyte-Bioreactors for the Ammonium Removal

An increased blood ammonium concentration (hyperammonemia) is toxic to the central nervous system, so removing excess ammonium from the bloodstream is an important task. One of the solutions may be the use of erythrocyte-bioreactors (EBRs) with ammonium-processing enzymes loaded inside. Earlier attempts have been made to create such EBRs based on glutamate dehydrogenase (GDH) or glutamine synthetase; however, these EBRs were not effective enough. We have previously shown that the reasons for this were the low permeability of the erythrocyte membrane for the substrates of these reactions (α-ketoglutarate and glutamate) and the low activity of the included GDH (due to its high molecular mass and tendency towards aggregation at an increase in concentration above 0.1 mg/mL), and also that the low membrane permeability problem for α-ketoglutarate and glutamate could be overcome if GDH and alanine aminotransferase were included into EBR together, because these metabolites should be consumed and reproduced within such EBR cyclically. However, the problem of low GDH activity remains if GDH is the main ammonium processing enzyme. To improve the efficiency of GDH incorporation into erythrocytes and the quality of the EBRs (their erythrocyte indices and osmotic fragility), these parameters were compared for various hypoosmotic methods for loading GDH into cells. In addition, a comparison was made of previously used GDH from bovine liver and GDH from Proteus sp. It was shown that the method of flow dialysis was the most effective and allowed the inclusion of the GDH amount 3 times exceeding the inclusion achieved during the hypoosmotic dialysis. The properties of erythrocytes after exposure to this method changed slightly. GDH from Proteus sp. did not aggregate with an increase in its concentration and thereby allowed an approximately 18-fold increase in the specific activity of the enzyme in erythrocytes. Thus, this GDH is a promising enzyme for creating effective EBRs for ammonium removing.

Comparative methodological studies of L-asparaginase encapsulation into erythrocytes

Background. L-asparaginase is an enzyme, widely used in the therapy of acute lymphoblastic leukemia in children and adults, but its use is limited due to a wide range of side effects and anaphylactic reactions. L-asparaginase loaded into erythrocytes can solve these problems. This enzyme is protected from the immune system and plasma proteases due to erythrocyte membrane, but continues to work inside the cell because its membrane is permeable to L-asparagine. Thus, the half-life of the drug increases and anaphylactic reactions reduce. The encapsulation of L-asparaginase into erythrocytes can be performed by various osmotic methods. Each of them is characterized by the amount of encapsulated enzyme, the cell yield, as well as by the quality indices of the survived erythrocytes. An important parameter of each method is the possibility to provide sterility of this dosage form for the clinical use.The aim of the study was the comparing of three osmotic methods of L-asparaginase encapsulation into erythrocytes (hypo-osmotic lysis, dialysis and flow dialysis) to select the most promising method for clinical use.Materials and methods. A suspension of erythrocytes of healthy donors (hematocrit 60–70%) was mixed with L-asparaginase from E. сoli. The procedures of hypotonic reversible lysis, dialysis in dialysis bags, or flow dialysis using pediatric dialyzers were performed. The physiological osmolality was restored in suspensions after the procedure by the addition of a hypertonic solution, and they were incubated for 30 min at 37 °C. Then the cells were washed in isotonic phosphate-buffered saline with pH 7.4. Activity of L-asparaginase, volume, hematocrit, hematological indices and osmotic cell fragility of erythrocytes were measured in the suspensions of erythrocytes before and after the enzyme encapsulation procedure.Results. An optimal osmolality of the hypotonic buffer for each method was selected and was equal to 90–110 mOsm/kg. The yields of encapsulation were 4.2 ± 2.0, 6.0 ± 2.3 and 16.2 ± 2.2 % for hypotonic lysis, dialysis and flow dialysis, respectively. The hematological indices of the obtained erythrocyte-carriers differed from the corresponding parameters of the initial erythrocytes, but did not differ significantly for different methods.Conclusion. Comparative investigation of mentioned above parameters allowed choosing the method of flow dialysis as the most promising for clinical use.

Rapid Elimination of Blood Alcohol Using Erythrocytes: Mathematical Modeling and In Vitro Study.

Erythrocytes (RBCs) loaded with alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALD) can metabolize plasma ethanol and acetaldehyde but with low efficiency. We investigated the rate-limiting factors in ethanol oxidation by these enzymes loaded into RBCs. Mathematical modeling and in vitro experiments on human RBCs loaded simultaneously with ADH and ALD (by hypoosmotic dialysis) were performed. The simulation showed that the rate of nicotinamide-adenine dinucleotide (NAD+) generation in RBC glycolysis, but not the activities of the loaded enzymes, is the rate-limiting step in external ethanol oxidation. The rate of oxidation could be increased if RBCs are supplemented by NAD+ and pyruvate. Our experimental data verified this theoretical conclusion. RBCs loaded with the complete system of ADH, ALD, NAD+, and pyruvate metabolized ethanol 20–40 times faster than reported in previous studies. The one-step procedure of hypoosmotic dialysis is the optimal method to encapsulate ADH and ALD in RBCs after cell recovery, encapsulation yield, osmotic resistance, and RBC-indexes. Consequently, transfusion of the RBCs loaded with the complete metabolic system, including ADH, ALD, pyruvate, and NAD+ in the patients with alcohol intoxication, may be a promising method for rapid detoxification of blood alcohol based on metabolism.

Erythrocytes as Carrier for Drugs, Enzymes and Peptides

Erythrocytes are potential biocompatible carriers for different drugs, peptide molecules and different enzymes. Now a day the method that is used for encapsulation of pharmaceuticals into the erythrocytes mainly based on the hypo-osmotic dialysis. Encapsulation of these drugs or enzymes or peptides into erythrocytes significantly changes the pharmacokinetic properties of drugs in both animals and humans, enhancing liver and spleen uptake and targeting the reticuloendothelial system. By the encapsulation of these into erythrocytes can be applied as targeted drug delivery systems. Erythrocytes are successful as carrier systems for different drugs, enzymes and peptide molecules. The result after using drug encapsulated in erythrocytes is more compared to the free form of the drug.

Preparation and in-vitro characterization of tramadol-loaded carrier erythrocytes for long-term intravenous delivery

The hypo-osmotic dialysis method was used for preparation of tramadol-loaded human intact erythrocytes. In response to rapid drug escape from the erythrocytes, a membrane cross-linker, glutaraldehyde, was used successfully. The resulting carrier cells were validated in terms of the accuracy and precision of the whole drug loading procedure. The average loaded amount, entrapment efficiency and cell recovery were 1.9041 mg, 95.98% and 85.13%, respectively. The effects of different drug concentrations on loading parameters were studied with the concentration of 10 mg/ml selected as optimal. A series of in-vitro characteristics of carrier erythrocytes, including tramadol release behaviour, haematological indices, particle size distribution, scanning electron microscopy, and osmotic/turbulence fragilities were determined compared with the sham-entrapped and unloaded cells. The results of these in-vitro tests indicated that the erythrocytes did not undergo remarkable irreversible size and shape/topology changes, but the fragility of the membranes of the processed cells were increased. The collective results of this study showed that the optimized method of entrapment was suitable for the encapsulation of tramadol in erythrocytes with the final carrier cells ready to enter the in-vivo animal studies as a promising long-circulating carrier for tramadol.

Study of the factors influencing the encapsulation of zidovudine in rat erythrocytes

Antiretroviral-loaded erythrocytes offer a promising therapy against HIV owing to their potential to deliver this kind of drugs to macrophages and reticulo-endothelial (RES) tissues. The aim of the present work was to develop and optimize a hypotonic dialysis method for the encapsulation of the antiretroviral Zidovudine (AZT) in rat erythrocytes. The influence of several factors in the encapsulation was also evaluated. Variables such as the initial AZT concentration, the dialysis time, and the dialysis bag/buffer volume ratio exhibited statistically significant differences in the encapsulation of the drug in erythrocytes. The amount of drug encapsulated was related to the different values of the variables by multiple linear regression. Osmotic fragility and haematological parameters were estimated as indicators of erythrocyte viability. No statistically significant differences in the osmotic fragility profiles of the control and carrier erythrocytes were observed, and this parameter was also independent of the dialysis concentration of AZT, the hypo-osmotic dialysis time, and the dialysis bag/buffer volume ratio. The in vitro release of AZT from carrier erythrocytes pointed to a fast leakage of the drug; however, around 30% of the drug remained encapsulated for a prolonged period of time. Pre-dialysis diamide treatment did not have a significant effect on the encapsulation and release of AZT in erythrocytes.

In vitro studies of amikacin-loaded human carrier erythrocytes

Erythrocyte-encapsulated antibiotics have the potential to provide an effective therapy against intracellular pathogens. The advantages over the administration of free antibiotics include a lower systemic dose, decreased toxicity, a sustained delivery of the antibiotic at higher concentrations to the intracellular site of pathogen replication, and increased efficacy. In this study, the encapsulation of amikacin by human carrier erythrocytes prepared using a hypo-osmotic dialysis was investigated. The effects of the initial amikacin dialysis concentration and hypo-osmotic dialysis time on the encapsulation efficiency of amikacin were determined, and the osmotic fragility and hematologic parameters of amikacin-loaded carrier erythrocytes were measured. The efficiency of amikacin entrapment by carrier erythrocytes was dependent on the initial dialysis concentration of the drug. Statistically significant differences in the osmotic fragility profiles between control and carrier erythrocytes were observed, which were dependent on the hypo-osmotic dialysis time and on the dialysis concentration of amikacin. Mean hematologic parameters were evaluated and compared with unloaded, native erythrocytes; the mean corpuscular volume (MCV) of amikacin-loaded carrier erythrocytes was statistically significant smaller. Amikacin demonstrated a sustained release from loaded erythrocytes over a 48-h period, which suggests a potential use of the erythrocyte as a slow systemic-release system for antibiotics.

A 9‐yr evaluation of carrier erythrocyte encapsulated adenosine deaminase (ADA) therapy in a patient with adult‐type ADA deficiency

Adenosine deaminase (ADA) deficiency is an inherited disorder which leads to elevated cellular levels of deoxyadenosine triphosphate (dATP) and systemic accumulation of its precursor, 2-deoxyadenosine. These metabolites impair lymphocyte function, and inactivate S-adenosylhomocysteine hydrolase (SAHH) respectively, leading to severe immunodeficiency. Enzyme replacement therapy with polyethylene glycol-conjugated ADA is available, but its efficacy is reduced by anti-ADA neutralising antibody formation. We report here carrier erythrocyte encapsulated native ADA therapy in an adult-type ADA deficient patient. Encapsulated enzyme is protected from antigenic responses and therapeutic activities are sustained. ADA-loaded autologous carrier erythrocytes were prepared using a hypo-osmotic dialysis procedure. Over a 9-yr period 225 treatment cycles were administered at 2-3 weekly intervals. Therapeutic efficacy was determined by monitoring immunological and metabolic parameters. After 9 yr of therapy, erythrocyte dATP concentration ranged between 24 and 44 micromol/L (diagnosis, 234) and SAHH activity between 1.69 and 2.29 nmol/h/mg haemoglobin (diagnosis, 0.34). Erythrocyte ADA activities were above the reference range of 40-100 nmol/h/mg haemoglobin (0 at diagnosis). Initial increases in absolute lymphocyte counts were not sustained; however, despite subnormal circulating CD20(+) cell numbers, serum immunoglobulin levels were normal. The patient tolerated the treatment well. The frequency of respiratory problems was reduced and the decline in the forced expiratory volume in 1 s and vital capacity reduced compared with the 4 yr preceding carrier erythrocyte therapy. Carrier erythrocyte-ADA therapy in an adult patient with ADA deficiency was shown to be metabolically and clinically effective.

Temperature-induced shape transformation of carrier erythrocytes

Erythrocytes have been proposed as cellular carriers for enzymes and drugs for protected delivery, slow release, or targeting to specific organs. Several studies have established the heterogeneity of resealed carrier erythrocytes concerning various characteristics, including morphology. Carrier erythrocytes were prepared by hypo-osmotic dialysis and consecutive iso-osmotic resealing at different temperatures, and the morphology of these cells was studied by scanning electron microscopy. Different cell shapes could be prepared by temperature variations during resealing or incubation. All intermediate shapes of echinocytosis were observed at low temperatures or with depleted energy supply but could be reversed to biconcave discocytes. Resealing at 4 degrees C resulted in highest percentage of echinocytes III, spherocytes, and leaky cells. Spontaneous resealing of 20% of the cells was obtained at high lysis temperatures. Combining low temperatures for lysis and high temperatures for resealing and sufficient energy supply are advantageous for highest recovery of biconcave discocytes. Shape of the erythrocyte, is the result of divergent forces, and temperature during resealing was found to be an important factor.

Preparation of lipid-free human hemoglobin by dialysis and ultrafiltration.

Dialysis of human red blood cells using a hypotonic solution and a commercial kidney dialysis unit followed by ultrafiltration through 0.1 micron pore hollow fibers provides an easily managed method for isolation of lipid-free hemoglobin. High pressure liquid chromatography analysis of lipid-free hemoglobin (LFHB) indicates 99-100% protein purity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that LFHB migrates as a single band. The process requires hypoosmotic dialysis of human RBC to a final 119-139 (av 132) mosmol/kg osmotic pressure. Additional reduction in osmotic pressure results in irreversible cell lysis which results in lipid contamination of the hemoglobin. Processing one-half liter of packed red blood cells requires 10 h, resulting in an average of 90% hemoglobin recovery.

Erythrocytes as carriers for heparin. Preliminary in vitro and animal studies.

Encapsulation of heparin into resealed carrier erythrocytes may be useful in the prevention of thromboembolism because heparin may be released locally during retraction of fresh thrombi. Heparin encapsulation in human and canine erythrocytes was achieved by hypo-osmotic dialysis with 44% and 36% encapsulation, respectively. Encapsulated heparin did not leak from carrier erythrocytes in vitro. In vivo survival of heparin-loaded carrier erythrocytes in dogs was biphasic: After an initial phase of cell loss the carrier cells survived in circulation with a half-life of 28 h. The present results seem to warrant clinical studies with heparin-loaded carrier erythrocytes.

In vivo clearance of antibody-sensitized human drug carrier erythrocytes.

Antibody coating of resealed drug carrier erythrocytes may be useful for drug targeting to the reticuloendothelial system. We have investigated the survival in the circulation of anti-Rh antibody (IgG anti-D)-coated autologous erythrocytes loaded with gentamicin by hypoosmotic dialysis. Five subjects were injected with 15.2 +/- 0.4 ml and five additional subjects with 62.8 +/- 1.5 ml carrier cells. Survival of the cells was monitored by intraerythrocytic gentamicin concentration in blood. In the first subject group initial t1/2 was 0.21 +/- 0.06 hours and terminal t1/2 was 1.71 +/- 0.36 hours. In the second group initial t1/2 was 0.59 +/- 0.21 hours followed by a slow phase with a t1/2 of 89 +/- 28 hours. Results indicate that rapid drug delivery to the reticuloendothelial system by antibody-sensitized carrier erythrocytes is possible, but small volumes of erythrocytes seem more efficient.

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-μm 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-poly-acrylamide 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.

Survival of gentamicin-loaded carrier erythrocytes in healthy human volunteers.

Resealed erythrocytes are potential slow release carriers for drugs and enzymes. We have investigated carrier erythrocyte survival in human volunteers using gentamicin (G) as encapsulated cell marker; G was readily incorporated into red cells by hypo-osmotic dialysis (87% efficiency of incorporation) and did not exit from carrier cells in vitro. Six healthy young volunteers were injected with 59 +/- 7 ml carrier erythrocytes containing 56 +/- 13 mg G. G levels were measured in plasma and haemolysed whole blood by RIA. After an initial phase of cell loss (up to 4.5 h post-injection) the carrier erythrocytes survived in circulation with a half-life of 22 days, as was indicated by intracellular G concentration. G levels were detectable in plasma during the first 90 min after injection. This indicates haemolysis of some carrier cells. In conclusion, carrier erythrocytes appear to circulate longer than any other drug carrier under investigation and may well serve as innocuous slow release system.

Release of vitamin B12 from carrier erythrocytes in vitro.

Resealed erythrocyte ghosts (carrier erythrocytes) are potential in vivo carriers for exogenous enzymes or drugs, but data on carrier erythrocyte survival and clearance rate in humans are not available. We have measured the in vitro efflux of vitamin B12 encapsulated in human red cell by hypo-osmotic dialysis, as a preliminary for its use as a marker for in vivo human studies. Vitamin B12 was encapsulated into erythrocytes at a relative incorporation efficiency of 60%. In vitro hemolysis of carrier erythrocytes was minimal over 40 h, but vitamin B12 was rapidly lost from the cells, efflux t/2 was 5 h, presumably by diffusion through the intact cell membrane. Vitamin B12 (Vit B12) may, nevertheless, be a suitable marker for short-term human studies on carrier erythrocyte splanchnic clearance.

Morphology of haemoglobin-containing human erythrocyte ‘ghosts’

Resealed erythrocyte ‘ghosts’ have been proposed as in vivo carriers for enzymes in the therapy of inherited metabolic diseases. A long life-span of this carrier is required when the erythrocyte ‘ghost’ is intended to be the site of substrate degradation in the circulation. Erythrocyte ‘ghosts’ have been prepared that show cell content and membrane transport characteristics that are closely similar to those of normal erythrocytes. Since the morphology of these ‘ghosts’ could probably also affect the in vivo life-span, haemoglobin-containing human erythrocyte ‘ghosts’ have been studied using scanning electron-microscopy. Preparation of the erythrocyte ‘ghosts’ involved a method of hypo-osmotic dialysis and consecutive iso-osmotic dialysis. Samples for scanning electron microscopy were prepared by fixation in glutaraldehyde, with post-fixation in osmium tetroxide. Dehydration was obtained by increasing concentrations of ethanol and critical-point-drying. Control experiments with normal erythrocytes showed no major artefacts by the methods used. Erythrocyte ‘ghosts’ showed polymorphic shapes. Two thirds were stomatocytes, but echinocytes and cells with deep invaginations or inter-twisted infoldings were also observed. Distribution of the different cell types could be affected by the preparation techniques used. Washing of the erythrocyte ‘ghosts’ at very low centrifugation speeds resulted in 60% of the cells appearing as biconcave discoids and one third as stomatocytes. The results demonstrate that the preparation disturbed the balance of the biconcave shape of resealed erythrocyte ‘ghosts’. Minor alterations of the methods allowed the preparation of erythrocyte ‘ghosts’, the majority of which showed morphology closely similar to that of normal erythrocytes. Attention must be given to all preparation factors to avoid irreversible damage and therefore prior to use of these cells in patients or when methods are altered, the morphology of these cellular carriers must be closely controlled.