Artificial red cells with crosslinked hemoglobin membranes

Artificial cells containing concentrated hemoglobin (Hb) solution were prepared by interfacial polymerization of Hb with glutaraldehyde (GA) in liquid membrane capsules (LMC). A solution containing 30% of Hb was emulsified in mineral oil as red cell-size microdroplets, and this emulsion was dispersed in an aqueous phase containing glutaraldehyde to form LMC. The LMC were semipermeable templates that held the microdroplets of Hb in suspension while GA diffused through the oil to the microdroplet surfaces. The GA crosslinked Hb at the surface of each microdroplet to form an artificial red-cell membrane encapsulating Hb solution. A water-soluble surfactant was used to eject the cells from the LMC and suspend them in saline. Several surfactants were evaluated. Cell size was controlled by agitation speed during preparation of the original emulsion. Cells of 2.52 = ±1.69 μm were prepared. The encapsulated Hb retained capacity to bind and release O₂. The cells had aP ₅₀ of 8.9 torr (1200 Pa) and a capacity of 0.55 cc O₂/g of total Hb, indicating that the crosslinked portion of the Hb did not contribute to O₂ transport.     

Blood‐Catalyzed RAFT Polymerization

The use of hemoglobin (Hb) contained within red blood cells to drive a controlled radical polymerization via a reversible addition‐fragmentation chain transfer (RAFT) process is reported for the first time. No pre‐treatment of the Hb or cells was required prior to their use as polymerization catalysts, indicating the potential for synthetic engineering in complex biological microenvironments without the need for ex vivo techniques. Owing to the naturally occurring prevalence of the reagents employed in the catalytic system (Hb and hydrogen peroxide), this approach may facilitate the development of new strategies for in vivo cell engineering with synthetic macromolecules.     

Preparation and in vitro characteristics of a blood substitute based on pyridoxylated polyhemoglobin

Stroma-free hemoglobin (SFHb) is prepared from outdated blood. Red cells are washed, lysed in hypotonic phosphate buffer, and stromal lipid is then removed by toluene extraction and high-speed centrifugation. Pyridoxal-phosphate (PP) added in a 4∶1 molar ratio to deoxygenated SFHb, is covelently linked across the polyphosphate binding site of the Hb tetramer by reduction with NaBH₄ under N₂ for 18 h. Excess reagents are removed by dialysis. Subsequent crosslinking using 5.0% glutaraldehyde in the presence of lysine for 12–36 h yields soluble, macromolecular polyhemoglobin (PolyHb). Progress of the reaction is monitored by gel chromatography. In vitro work shows that different molecular weight (MW) PolyHb can be produced, ranging in weight from 130,000 to over 1,000,000. Intermediate MW PolyHb (<600,000) remains stable for months at 4°C, withstands prolonged incubation at 37°C, and has a viscosity similar to blood. SFHb has a lowP ₅₀=15 torr, which has been raised to 26 torr by pyridoxylation. When PP-SFHb is crosslinked into PP-PolyHb, it can reversibly carry oxygen with a lower affinity (P ₅₀=16 torr) than nonpyridoxylated PolyHb (P ₅₀=11 torr). These favorable physicochemical properties of PP-PolyHb suggest that further progress toward a potential artificial blood substitute has been made.     

基于微流控芯片电泳化学发光检测人单个红细胞中血红蛋白的含量

运用自行设计组装的微流控芯片电泳化学发光检测装置和单细胞分析专用玻璃微流控芯片,建立了一种测定人单个血红细胞中血红蛋白(Hb)含量的新方法。该方法采用双T型的窄进样通道,宽反应通道及适中分离通道的玻璃微流控芯片,集成单个细胞的进样、固定、溶胞、分离和检测等操作于一块微流控芯片上。以p H 10.5的硼砂缓冲液为电泳介质,选用鲁米诺-过氧化氢化学发光体系,对人单个红细胞中血红蛋白的含量进行测定。血红蛋白的质量在2.0~90 pg范围内,与化学发光强度(峰高)呈良好的线性关系,检出限(S/N=3)为0.8 pg。通过对19个血红细胞进行检测,得到人单个血红细胞中血红蛋白的含量在14~68 pg范围内,该结果与无氰HGB测量法测得的总体细胞血红蛋白的平均值(34.5 pg)基本一致。     

A blood substitute from hydroxyethyl starch and hemoglobin

One method to increase the retention time of hemoglobin (Hgb) is to react it with a hydroxyethyl starch (HES) molecule. To examine this hypothesis, polymer-bound hemoglobin compounds were synthesized by the dialdehyde route. The electrophoretic mobility patterns indicate complete binding of the Hgb. Preliminary exchange-transfusion experiments in rates showed that they could survive for at least 10 h at Hct<10% when transfused with 6% HES-Hgb solutions. The retention time of the Hgb in the urine was increased to 12 h with these new polymers.     

Selection and microencapsulation of an “NADH-oxidizing” bacterium and its use for NAD regeneration

An alternative approach to the regeneration of coenzymes is described here using immobilized microorganisms possessing “NADH-oxidase” function. Bacteria containing NADH-oxidase activity are immobilized by microencapsulation within artificial cells. In this form, the microencapsulated bacteria can recycle NADH back to NAD in the presence of molecular oxygen as an electron acceptor. The only byproduct of the recycling reaction is water. In order to perform the biological regeneration of NAD, the activity of NADH-oxidase was investigated in 13 strains of aerobic bacteria and yeast. The NADH-oxidizing bacteriaLeuconostoc mesenteroides exhibited the highest activity among the microorganisms tested. The permeabilized bacteria showed 10% of their initial activity after microencapsulation. Light and electron microscopy studies of bacteria loaded microcapsules have been done. Enzymatic properties of microcapsule-immobilized bacteria were investigated in comparison with those of the free enzyme complex.Leuconostoc mesenteroides, containing NADH-oxidase, has been microencapsulated together with 3α-hydroxysteroid dehydrogenase (3α-HSDH) for stereospecific steroid oxidation. In a batch reactor, 2 mg of NAD, with recycling, allowed the same substrate consumption as 4.4 mg of NAD without recycling. The microencapsulated system can be used repeatedly. The system is functional for 10 h, during which time each molecule of NAD has been used 7.6 times.     

人工氧载体研究进展

血液需求的激增和异体输血的不安全性等问题的出现,促使人们合成“血液替代品”。通过对天然氧载体(即血红蛋白)结构与性能的清晰认识,已有多种人工氧载体被成功合成,并应用于临床试验。人工氧载体可分为全氟碳化合物、血红蛋白基氧载体、合成血红素及其高分子配合物三大类。全氟碳化合物虽大部分已退出人工血液市场,但因其具有治疗作用,研究工作仍在进行。为了降低血红蛋白基氧载体的副作用,已采用多种方法对血红蛋白进行改性,如采用化学修饰、微囊包裹(HbV)、重组和仿生纳米等技术。其中,血红蛋白囊泡模拟红细胞的结构,其粒径相对较大(250nm),副作用相对较低,是目前血红蛋白基氧载体的发展趋势。人工合成血红素如栏式卟啉只溶于有机溶剂,为增加其水溶性,可使其与白蛋白、木糖醇酶和环糊精等高分子结合为配合物,经动物实验表明,这些高分子金属配合物在体内具有运送氧气功能。除主要在临床上用作血液代替品外,人工氧载体还在肿瘤治疗、器官移植和缺血/再灌注损伤的预防等方面具有重要的临床应用价值。     

SEM observation of the live morphology of human red blood cells under high vacuum conditions using a novel RTIL

In this study, we succeed in visualizing a ‘living’ morphology of red blood cells (RBCs) by a rapid and simple scanning electron microscope (SEM) pretreatment using a hydrophilic room temperature ionic liquid (RTIL) with asymmetrical structure. The conventional pretreatment induces size shrinkage of the cells, and the diameter observed in an SEM (less than 6 µm) was smaller than that in an optical microscope (8 µm). Though RBCs are well‐known to be easily deformed with the environmental conditions, the cells did not show the deformation when they were pretreated with our RTILs. Since the chemical structure of our RTIL resembles a ‘choline’, which is a typical bioactive material, and has biocompatibilities, they may make it avoid from the deformation of RBCs. In fact, only immersing RBCs, our novel RTIL can provide a clear SEM image without size shrinkage and deformation. Interestingly, the obtained SEM image shows 8 µm in the diameter, almost same size as that in optical microscope. Therefore, this method is expected for novel SEM pretreatment for various biological samples observation as ‘living’ matter. Copyright © 2014 John Wiley & Sons, Ltd.     

Electrochemical Red Blood Cell Counting: One at a Time

We demonstrate that the concentration of a red blood cell solution under physiological conditions can be determined by electrochemical voltammetry. The magnitude of the oxygen reduction currents produced at an edge‐plane pyrolytic graphite electrode was diagnosed analytically at concentrations suitable for a point‐of‐care test device. The currents could be further enhanced when the solution of red blood cells was exposed to hydrogen peroxide. We show that the enhanced signal can be used to detect red blood cells at a single entity level. The method presented relies on the catalytic activity of red blood cells towards hydrogen peroxide and on surface‐induced haemolysis. Each single cell activity is expressed as current spikes decaying within a few seconds back to the background current. The frequency of such current spikes is proportional to the concentration of cells in solution.     

The Redox Potential of the β-93-Cysteine Thiol Group in Human Hemoglobin Estimated from In Vitro Oxidant Challenge Experiments

Glutathionyl hemoglobin is a minor form of hemoglobin with intriguing properties. The measurement of the redox potential of its reactive β-⁹³-Cysteine is useful to improve understanding of the response of erythrocytes to transient and chronic conditions of oxidative stress, where the level of glutathionyl hemoglobin is increased. An independent literature experiment describes the recovery of human erythrocytes exposed to an oxidant burst by measuring glutathione, glutathione disulfide and glutathionyl hemoglobin in a two-hour period. This article calculates a value for the redox potential E₀ of the β-⁹³-Cysteine, considering the erythrocyte as a closed system at equilibrium described by the Nernst equation and using the measurements of the literature experiment. The obtained value of E₀ of −121 mV at pH 7.4 places hemoglobin as the most oxidizing thiol of the erythrocyte. By using as synthetic indicators of the concentrations the electrochemical potentials of the two main redox pairs in the erythrocytes, those of glutathione–glutathione disulfide and of glutathionyl–hemoglobin, the mechanism of the recovery phase can be hypothesized. Hemoglobin acts as the redox buffer that scavenges oxidized glutathione in the oxidative phase and releases it in the recovery phase, by acting as the substrate of the NAD(P)H-cofactored enzymes.     

活体小鼠中单个红细胞的拉曼光谱分析

应用拉曼光谱与光镊技术对活体小鼠毛细动脉和静脉血管中的全血以及毛细血管中的红细胞和体外的全血及单个红细胞进行研究;光谱分析显示,可以获得活体血液的拉曼光谱;在动脉毛细管和静脉毛细管中可以观察到清晰的携氧与去氧血液的不同光谱;体内血管中血红细胞的血红蛋白浓度比体外的更高,在动脉毛细管中的血红细胞的携氧态特征峰明显,且具有...     

一种全合成型人工红血球的研究进展

一种全合成型人工红血球的研究进展;人工红血球;全合成型白蛋白－血红素载体;氧气输送;安全性;综述     

Improved HPLC method for the determination of ribavirin concentration in red blood cells and its application in patients with COVID-19

Ribavirin is a synthetic, broad-spectrum antiviral drug. Ribavirin is recommended as an antiviral drug in the Interim Guidance for Diagnosis and Treatment (the seventh edition) of COVID-19. The ribavirin levels in red blood cells may be closely related to both its efficacy and adverse drug reactions. In this study, a simple and fast HPLC–UV method was established to determine the concentrations of total ribavirin in the red blood cells of 13 patients with COVID-19. Phosphorylated ribavirin was dephosphorylated by phosphatase incubation to obtain the total amount of ribavirin in red blood cells. The chromatographic column was an Atlantis C₁₈. The recoveries were 85.45–89.05% at three levels. A good linear response was from 1 to 200 μg/ml, with a correlation coefficient of r² = 0.9991. The concentration of total ribavirin in the red blood cells of the patients ranged from 30.83 to 133.34 μg/ml. The same samples without phosphatase incubation ranged from 4.07 to 20.84 μg/ml. About 85% of ribavirin was phosphorylated in red blood cells. In addition, we observed changes in these patients' hematological parameters and found that the erythrocyte, hemoglobin and hematocrit declined to the lowest levels on the fifth day after discontinuation of ribavirin (p < 0.05).     

Microencapsulation by a complex coacervation process using acid-precursor gelatin

This report describes our studies on the optimal conditions for the complex coacervation of an acid-precusor gelatin with an isoelectric point of pH 8.3 at high colloid concentration (5%). The following factors were studied: the amount of gelatin deposited and the electrical behavior of colloids.     

Characteristics and function of human hemoglobin vesicles as an oxygen carrier

A liposome‐encapsulated human hemoglobin (Neo Red Cell, (NRC) has been developed and evaluated as an artificial oxygen carrier. The NRC is a liposome‐encapsulated highly concentrated (>45%) stroma‐free human hemoglobin with inositol hexaphosphate (IHP as an allosteric effector), a coenzyme and substrates for reducing methemoglobin (metHb). The NRC's surface was coated with polyethylene glycol to prevent aggregation in plasma and to prolong their retention time in the blood stream. The oxygen binding behavior of the NRC in vitro was investigated and it was found that it effectively transports oxygen in vivo as an oxygen carrier. The oxygen binding behavior and kinetics were studied by the stopped‐flow method and the oxygen binding curve of the NRC was determined. The oxygen binding speed and binding coefficient (K_on) of NRC, washed human red blood cells (WRBC) and stroma‐free human hemoglobin (SFHb) were measured by stopped‐flow method. The oxygen binding speed of SFHb was the highest, while that of RBC was the lowest and that of NRC was intermediate. The oxygen binding of NRC ended within 60 msec when deoxy‐NRC was mixed with oxygen. The K_on of NRC was 2.9 × 10⁵, 10 times faster than that of RBC. The oxygen binding curve and P₅₀O₂ of NRC that contained various IHP concentrations were measured. The oxygen‐binding curve of the NRC sequentially shifted to the right as the IHP content was increased. Exchange transfusion of 70% was carried out for rats with NRC containing various concentrations of IHP and of Hb, and investigated the optimum concentration of NRC in vivo. The lactate value after exchange transfusion was three times higher than before exchange transfusion, when rats were subjected to exchange transfused with NRC that did not contain IHP. But the increase of lactate was suppressed when rats were transfused with NRC that contained IHP. When the Hb concentration of NRC was 5 and 6%, exchange transfused rats recovered to normality just like rats transfused with RBC. Copyright © 2000 John Wiley & Sons, Ltd.     

Dielectric characterization of Babesia bovis using the dielectrophoretic crossover frequency

Coinfection with the tick-transmitted pathogen Babesia spp. is becoming a serious health problem because of the erythrocyte invasion through Ixodes scapularis tick. The transmission of this protozoan by blood transfusion often results in high morbidity and mortality in recipients. A novel way to detect parasitized erythrocytes is by utilizing dielectrophoresis, an electrokinetic technique on a microfluidic platform, to improve the diagnostics of Babesia spp. The differences in the dielectric properties of Babesia spp.–infected erythrocytes versus healthy erythrocytes were exploited to design a fast and cost-effective diagnostic tool. One crucial factor for a successful diagnostic platform via dielectrophoretic separation is the dielectric characterization of Babesia-infected erythrocytes, which is investigated in this paper. The influence of medium conductivity and erythrocytes phenotype and genotype over the first crossover frequency (f_co1) are used to quantify the dielectric properties of the infected cells. A sigmoidal curve was plotted via curve fitting of the single-shell model, which has been proven appropriate for parasitized cell populations where considerable cell geometry variation occurs. The difference in these curves is relevant for the separation of cells population. Microliters of sample and reagent were used throughout this experiment; the scale, results obtained, and simplicity of the system often make it very suitable for point-of-care babesiosis disease diagnostics.     

Dielectrophoretic detection of electrical property changes of stored human red blood cells

The ability to transport and store a large human blood inventory for transfusions is an essential requirement for medical institutions. Thus, there is an important need for rapid and low-cost characterization tools for analyzing the properties of human red blood cells (RBCs) while in storage. In this study, we investigate the ability to use dielectrophoresis (DEP) for measuring the storage-induced changes in RBC electrical properties. Fresh human blood was collected, suspended in K2-EDTA anticoagulant, and stored in a blood bank refrigerator for a period of 20 days. Cells were removed from storage at 5-day intervals and subjected to a glutaraldehyde crosslinking reaction to “freeze” cells at their ionic equilibrium at that point in time and prevent ion leakage during DEP analysis. The DEP behavior of RBCs was analyzed in a high permittivity DEP buffer using a three-dimensional DEP chip (3DEP) and also compared to measurements taken with a 2D quadrupole electrode array. The DEP analysis confirms that RBC electrical property changes occur during storage and are only discernable with the use of the cell crosslinking reaction above a glutaraldehyde fixation concentration of 1.0 w/v%. In particular, cytoplasm conductivity was observed to decrease by more than 75% while the RBC membrane conductance was observed to increase by more than 1000% over a period of 20 days. These results show that the presented combination of chemical crosslinking and DEP can be used as rapid characterization tool for monitoring electrical properties changes of human RBCs while subjected to refrigeration in blood bank storage.     

火焰原子吸收光谱法测定人血红细胞中锌、铁、镁

人血红细胞样品经低渗Triton X-100溶液超声振荡溶血处理并稀释100倍后采用火焰原子吸收光谱法测定其中锌、铁和镁的含量。锌、铁和镁的检出限(3S/N)分别为2.44×10~(-3),2.85×10~(-2),1.56×10~(-3)mg·L~(-1),相对标准偏差(n=7)分别为0.69%～1.03%,2.85%～3.84%,0.92%～1.22%。应用该法分析同一混合人血红细胞样品,测定结果与酸消化法测得的结果一致,3种元素的加标回收率为95.0%～110.0%之间。