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Hemoglobin-Vesicles as Oxygen Carriers

Influence on Phagocytic Activity and Histopathological Changes in Reticuloendothelial System
      Hemoglobin-vesicles (HbV) have been developed for use as artificial oxygen carriers (particle diameter, 250 nm) in which a purified Hb solution is encapsulated with a phospholipid bilayer membrane. The influence of HbV on the reticuloendothelial system was studied by carbon clearance measurements and histopathological examination. The HbV suspension ([Hb] = 10 g/dl) was intravenously infused in male Wistar rats at dose rates of 10 and 20 ml/kg, and the phagocytic activity was measured by monitoring the rate of carbon clearance at 8 hours and at 1, 3, 7, and 14 days after infusion. The phagocytic activity transiently decreased one day after infusion by about 40%, but it recovered and was enhanced at 3 days, showing a maximum of about twice the quiescent level at 7 days, and then returned to the normal value at 14 days. The initial transient decreased activity indicates a partly, but not completely, suppressed defensive function of the body. The succeeding increased phagocytic activity corresponds to the increased metabolism of HbV. The histopathological examination with anti-human Hb antibody, hematoxylin/eosin, and oil red O stainings showed that HbV was metabolized within 7 days. Hemosiderin was very slightly confirmed with Berlin blue staining at 3 and 7 days in liver and spleen, though they completely disappeared at 14 days, indicating that the heme metabolism, excretion or recycling of iron proceeded smoothly and iron deposition was minimal. Electron microscopic examination of the spleen and liver tissues clearly demonstrated the particles of HbV with a diameter of about 1/40 of red blood cells in capillaries, and in phagosomes as entrapped in the spleen macrophages and Kupffer cells one day after infusion. The vesicular structure could not be observed at 7 days. Even though the infusion of HbV modified the phagocytic activity for 2 weeks, it does not seem to cause any irreversible damage to the phagocytic organs. These results offer important information for evaluating the safety issues of HbV for clinical use.
      Phospholipid vesicles encapsulating concentrated human hemoglobin (Hb, Hb vesicles, HbV) can serve as an oxygen carrier with sufficient oxygen transporting ability comparable to blood.
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      In vivo biodistribution of a radiorabeled blood substitute: 99mTc-labeled liposome-encapsulated hemoglobin in an anesthetized rabbit.
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      Physiologic responses to exchange transfusion with hemoglobin vesicles as an artificial oxygen carrier in anesthetized rats: changes in mean arterial pressure and renal cortical tissue oxygen tension.
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      Microvascular responses to hemodilution with Hb vesicles as red blood cell substitutes: influence of O2 affinity.
      The advantages of Hb-based oxygen carriers are the absence of a blood type antigen and infectious virus, a small particle size for the penetration through constricted vessels where red blood cells cannot penetrate, and stability for long-term storage.
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      Polyethyleneglycol-conjugation and deoxygenation enable long-term preservation of hemoglobin-vesicles as oxygen carriers in a liquid state.
      These characteristics will make it possible to use the oxygen carriers both in elective and emergency situations without blood type matching and virus tests. In this sense, infusion of oxygen carriers becomes superior to the conventional blood transfusion which still has the potential of mismatching, infection such as HIV and hepatitis virus, and graft-versus-host disease, and the problems of a short period of preservation. Even though the acellular Hb modifications, including polymerized Hb and polymer-conjugated Hb, are now undergoing the final stages of clinical trials,
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      the cellular structure of HbV (particle diameter, ∼ 250 nm) most closely mimics the characteristics of natural red blood cells such as the cell membrane function of physically preventing direct contact of Hb with the components of blood and vasculature during circulation. In comparison with some acellular Hb modifications, Hb encapsulation in vesicles not only prolongs the circulation time, but also suppresses hypertension induced by vasoconstriction; a theory that is suggested to be due to the high affinity of Hb with nitric oxide and carbon monoxide as vasorelaxation factors.
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      Free Hb released from RBC is rapidly bound to haptoglobin and removed from the circulation by hepatocytes. However, when the Hb concentration exceeds the haptoglobin binding capacity, unbound Hb is filtered through the kidney where it is actively absorbed. When the reabsorption capacity of the kidney is exceeded, hemoglobinuria and eventually renal failure occur.
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      Elimination of pyridoxalated polyhemoglobin after partial exchange transfusion in chimpanzees.
      The encapsulation of Hb completely suppresses renal excretion, though HbV particles as well as phospholipid vesicles (liposomes) or oil emulsions in the blood stream are finally captured by phagocytes in the reticuloendothelial system (RES, or mononuclear phagocytic system, MPS).
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      Physiological responses, organ distribution, and circulation kinetics in anesthetized rats after hypovolemic exchange transfusion with technetium-99m-labeled liposome-encapsulated hemoglobin.
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      A chemically modified (pyridoxalated) Hb was also reported to be captured by RES.
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      The main function of RES is protective elimination of foreign materials such as viruses, bacteria, metastatic tumor cells, and bacterial endotoxins.
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      It has been anticipated that the accumulation of phospholipid vesicles in phagocytic cells may lead to impairment or even blockade of their function.
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      Acute toxicity and depression of phagocytosis in vivo by liposomes: influence of lysophosphatidyl choline.
      This may cause adverse effects such as decreased resistance to infection, the metastatic spread of tumors, or spillover of endotoxins from the gut. Clinically approved Fluosol-DA (Green Cross Co., Osaka, Japan) as an oxygen carrier and lipid emulsion as a nutrient are trapped by the phagocytic cells.
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      Intralipid adversely affects reticuloendothelial bacterial clearance.
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      Effect of pyridoxalated stabilized stroma-free hemoglobin solution on the clearance of intravascular lipid by the reticuloendothelial system.
      Several reports measured the phagocytic activity by the carbon clearance method and clarified that the phagocytic activity is transiently depressed after the infusion and then recovered and enhanced.
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      Antineoplastic activity of sterically stabilized alkylphosphocholine liposomes in human breast carcinomas.
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      Influence of polymerizable phospholipid vesicels encapsulated hemoglobin on the murine immune system.
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      Biodistribution of the neo red cell and effect on the phagocytic activity of the reticuloendothelial system.
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      Influence of Fluosol-DA 20% on reticuloendothelial systems.
      The advantages of our HbV over the conventional Hb vesicles
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      Use of oxygen-15 to measure oxygen carrying capacity of blood substitutes in vivo..
      are a high encapsulation efficiency of Hb in phospholipid vesicles thus the total amount of lipids is significantly reduced,
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      • Tsuchida E
      Layer-controlled hemoglobin vesicles by interaction of hemoglobin with a phospholipid assembly.
      • Sakai H
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      Physical properties of hemoglobin vesicles as red cell substitutes.
      the surface modification of HbV with poly(ethylene glycol) that allows better hemodynamics due to the suppression of intervesicular aggregation and reduced viscosity,
      • Yoshioka H
      Surface modification of hemoglobin-containing liposomes with polyethylene glycol prevents liposome aggregation in blood plasma.
      • Sakai H
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      Surface-modification of hemoglobin vesicles with polyethyleneglycol and effects on aggregation, viscosity, and blood flow during 90%-exchange transfusion in anesthetized rats.
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      • Tsai AG
      • Kerger H
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      • Tsuchida E
      • Intaglietta M
      Subcutaneous microvascular responses to hemodilution with red cell substitutes consisting of polyethyleneglycol-modified vesicles encapsulating hemoglobin.
      reduced complement activation due to the suppression of interaction with plasma proteins,
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      Inhibition of liposome-induced complement activation by incorporated poly(ethylene glycol)-lipids.
      and longer circulation time and a moderate rate of entrapment and metabolism.
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      • Rudolph AS
      • Cliff R
      • Kwasoborski V
      • Goins BA
      Polyethyleneglycol-modified liposome-encapsulated hemoglobin: a long circulating red cell substitute.
      We thought these revised characteristics may be effective to maintain microcirculation and to reduce the burden on RES. In this study, the effect of HbV infusion on the RES function was analyzed by the carbon clearance measurement,
      • Arndt D
      • Zeisig R
      • Eue I
      • Sternberg B
      • Fichtner I
      Antineoplastic activity of sterically stabilized alkylphosphocholine liposomes in human breast carcinomas.
      • Hanada H
      • Kubo T
      • Ikeda M
      • Watanabe M
      Influence of Fluosol-DA 20% on reticuloendothelial systems.
      • van Etten EWM
      • ten Kate MT
      • Snijders SV
      • Bakker-Woudenberg IAJM
      Administration of liposomal agents and blood clearance capacity of the mononuclear phagocyte system.
      and also its metabolism and the influence on the tissue parenchymal cells was confirmed by histopathological examination.

      Materials and Methods

      Preparation of poly(ethylene glycol)-modified Hb-vesicles (HbV) polyethylene glycol (PEG)-modified HbV was performed at Waseda University under sterile conditions as previously reported in the literature.
      • Sakai H
      • Hara H
      • Yuasa M
      • Tsai AG
      • Takeoka S
      • Tsuchida E
      • Intaglietta M
      Molecular dimensions of Hb-based O2 carriers determine constriction of resistance arteries and hypertension in conscious hamster model.
      • Takeoka S
      • Ohgushi T
      • Terase K
      • Ohmori T
      • Tsuchida E
      Layer-controlled hemoglobin vesicles by interaction of hemoglobin with a phospholipid assembly.
      • Sakai H
      • Takeoka S
      • Park SI
      • Kose T
      • Izumi Y
      • Yoshizu A
      • Nishide H
      • Kobayashi K
      • Tsuchida E
      Surface-modification of hemoglobin vesicles with polyethyleneglycol and effects on aggregation, viscosity, and blood flow during 90%-exchange transfusion in anesthetized rats.
      Hb was purified from outdated donated blood provided by the Hokkaido Red Cross Blood Center (Sapporo, Japan). The encapsulated carbonylhemoglobin (HbCO, 38 g/dl) contained 5.9 mmol/L of pyridoxal 5′-phosphate (PLP, Merck, Whitehouse Station, NJ) as an allosteric effector at a molar ratio of Hb/PLP = 3, and 5 mmol/L of homocysteine (Aldrich, Milwaukee, WI) as a reductant. The lipid bilayer was composed of Presome PPG-I [a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, cholesterol, and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol at a molar ratio of 5/5/1 (Nippon Fine Chemicals, Osaka, Japan)]. The HbCO solution and the lipids were mixed and stirred for 12 hours at 4°C. The resulting mutilamellar vesicles were extruded through membrane filters using RemolinoTM (Millipore, Bedford, MA) with a final filter pore size of 0.22 μm. After rinsing with saline, the HbV surface was modified with PEG (molecular weight 5 kd, 0.3 mol % to the outer surface of lipids) using 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-PEG (Sunbright DSPE-50H, H salt type, NOF Co., Tokyo, Japan), where succinic acid is a crosslinker between PEG and DSPE. The hydrophobic alkyl chains of PEG-DSPE are inserted into the lipid bilayer of HbV by mixing the HbV suspension with a saline suspension at 37°C for 2 hours. After decarbonylation of HbCO to HbO2, the resulting PEG-modified HbV was ultracentrifuged to remove the unintroduced PEG-lipid, and redispersed in saline at the Hb concentration of 10 g/dl. The suspension was then filtered through sterilizable filters (pore size: 0.45 μm). The physicochemical parameters of the HbV are as follows: particle diameter, 251 ± 80 nm; [Hb], 10 g/dl; [metHb], <5%; [HbCO], < 3%; phospholipids, 4.0 g/dl; cholesterol, 1.7 g/dl; and oxygen affinity (P50), 32 Torr.

      Injection of Sample Suspensions

      All animal studies were approved by the Animal Subject Committee of Keio University School of Medicine and performed according to NIH guidelines for the care and use of laboratory animals (NIH publication 85–23 Rev. 1985).
      Experiments were carried out using 70 male Wistar rats (200–210 g, Charles River Co., Tokyo, Japan). They were anesthetized with diethylether, and the sample suspension was infused into the tail vein. The sample was either HbV (10 ml/kg, n = 15; 20 ml/kg, n = 19) or saline (20 ml/kg, n = 15) and 10 wt% of IntralipidTM suspension (Pharmacia, Stockholm, Sweden) 20 ml/kg, n = 15). Six animals were used to obtain the control values. All of the rats were housed in cages and provided with food and water ad libitum in a temperature controlled room on a 12 hour dark/light cycle.

      Carbon Clearance Measurement

      After 8 hours and 1, 3, 7, and 14 days, the rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (∼ 100 mg/kg body weight, Abbott Lab., North Chicago, IL). Polyethylene tubes (PE-50, Natsume Co., Tokyo) were implanted in the jugular vein. A carbon particle solution (Fount India Ink, Pelikan Co., Hannover, Germany) was diluted to 16 mg/ml with saline and infused at 10 ml/kg within 1 minute. The pink-colored rat skin immediately turned to black, indicating that the carbon particles were circulating throughout the body. Four, 10, and 20 minutes later, about 120 μl of blood was withdrawn from the vein, and exactly 50 μl of blood was diluted with 5 ml of a 0.1% sodium bicarbonate solution in a cuvette for spectrophotometer. Absorption at 675 nm was measured with the spectrophotometer (UV-2000, Shimadzu Co., Tokyo, Japan). The control blood was also measured before infusing the carbon particle solution. The phagocyte index (K) was calculated with the equation: K = 1/(t2t1) × ln(C1/C2) where C1 and C2 are the concentrations (absorbance) at time t1 and t2 (minutes), respectively. After the experiment, the animals were laparotomized to be sacrificed with acute bleeding from the abdominal aorta and to obtain the liver, spleen, and kidney, and then the lung and heart were resected en bloc for a histopathological study. The organs were soaked in 10% formalin immediately after the resection.

      Histopathological Study

      Paraffin sections were prepared from the 10% formalin-fixed organs, and stained with hematoxylin/eosin, anti-human Hb antibody, Berlin blue, and oil red O stainings. The human Hb in the HbV particles in the tissue was confirmed by staining with a rabbit polyclonal antibody against human Hb (DAKO A/S, Copenhagen, Denmark) as the primary antibody. This antibody does not cross-react with rat hemoglobin (which was evident from the result that rat red blood cells were not stained). Reaction with the second antibody and color development were performed with the Ventana alkaline phosphatase RED detection kit using the Ventana NX system (Ventana Med. System, Inc., Tucson, AZ). The percentage of the stained area was calculated with a computer software (IPLab, Fairfax, VA). The presence and location of hemosiderin including free irons released by the metabolism of heme were confirmed by Berlin blue stain. The neutral lipid deposition, which might be generated during the metabolism of the phospholipid components of the bilayer membrane of HbV, were examined by oil red O staining of the sliced organ specimens directly prepared from the formalin-fixed organs.
      To visualize the morphological changes in the HbV particles in the spleen and liver, transmission electron microscopic observation (with a high magnification) was performed. The spleen and liver, taken from the rat without carbon particle infusion, were cut in about 2 mm3 portions in 2.5% glutaraldehyde solution and then stored in 8% sucrose solution (0.1 mol/L phosphate buffer, pH 7.4). The fixed organs were then washed with 0.1 mol/L phosphate buffer, and stained with 2% osumic acid solution at 4°C for 2 hours. Next, the organs were first dehydrated with ethanol solution by stepwise increases in the ethanol content (50, 60, 70, 80, 90, 95, and 100%) for 10 minutes during each step, washed with propylene oxide, and then polymerized using Quetol 812 at 60°C for 28 hours. The obtained samples were sliced into 60 to70-nm sections by using an Ultracut S microtome. The sliced samples were stained with 3% uranyl acetate solution for 16 to 20 minutes and then treated with Satoh's lead solution (lead acetate, lead nitrate, and lead citrate) in citrate for 5 minutes, washed, and dried. The sample was observed and a picture taken with a transmission electron microscope (TEM, JEM-100CX, JEOL, Tokyo, Japan).

      Blood Serum Clinical Chemistry

      The rats receiving HbV but not carbon particles were used to analyze blood serum clinical chemistry (n = 15). After 8 hours and 1, 3, 7 and 14 days, the rats were anesthetized with an intraperitoneal injection of sodium pentobarbital. Polyethylene tubes (PE-50) were implanted in the carotid artery and about 4 ml of blood was withdrawn in heparinized syringe. Since HbV particles interfere with some analytes of serum clinical chemistry, the blood was ultracentrifuged (50,000 × g for 20 minutes) to completely remove the HbV particles in advance. The serum samples were stored in a refrigerator (−80°C) until the analyses. To evaluate function of liver as one of the main organs for the HbV metabolism, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were selected as the analytes (BML Inc., Kawagoe, Japan).

      Data Analysis

      Differences between the treatment groups were analyzed using a one-way analysis of variance followed by Fisher's protected least significant difference (PLSD) test. A paired t-test was used to compare the time dependent changes within each group. The changes were considered statistically significant if P < 0.05.

      Results

      Body and Spleen Weights

      All of the rats tolerated the overdose of the sample solutions. The original body weights were 210 to 225 g on average (Figure 1a). The body weight decreased, especially in the 20 ml/kg HbV group, by about 10% one day after the HbV administration. However, the body weight of the HbV groups returned to that of the control groups within 3 days and they grew normally.
      Figure thumbnail gr1
      Figure 1Changes in body weight (a) and spleen weight (b) after infusion of HbV (10 and 20 ml/kg), saline and Intralipid as controls. The values are mean ± SD. *Significantly different from the saline group (P < 0.05).
      The two HbV groups showed splenomegaly (Figure 1b). The spleen weight increased for the 20 ml/kg HbV group by about 500 mg, which is about 70% of the infused amount of HbV (3300 mg/kg × 0.21 kg = 693 mg), and the spleen weight tended to remain even after 2 weeks. The 10 ml/kg HbV group showed a spleen weight increase at 1 week and tended to decrease at 2 weeks.

      Changes in Phagocytic Activity

      The phagocytic index (K) dropped at 8 hours or 1 day after the HbV infusion by about 30 to 50%, though it never completely saturated (Figure 2). In the case of the 10 ml/kg HbV group, the K value recovered 3 days later and showed a maximum value at 1 week, and then ceased at 2 weeks. For the 20 ml/kg HbV infusion, the significantly high value of K, about twice the baseline value, was observed at 1 week, and then it ceased at 2 weeks. Thus the dramatic changes in the phagocytic activities were not irreversible. The changes in K for the saline and lipid microsphere groups were minimal.
      Figure thumbnail gr2
      Figure 2Changes in phagocytic activity after infusion of HbV (10 and 20 ml/kg), saline and Intralipid as controls. The values are mean ± SD. The phagocytic activity was obtained with the carbon clearance measurement. *Significantly different from the basal value, K = 0.053 ± 0.011 (P < 0.05).

      Histopathological Examination by Staining

      The histopathological examination of the spleen and liver after the 20 ml/kg infusion is shown in Figure 3. The human Hb in HbV particles were stained as red-colored portions with anti-human Hb antibody as a primary antibody. It was confirmed in advance with smears of human and rat blood and HbV suspension that the antibody reacts with only human Hb but not with rat Hb.
      Figure thumbnail gr3
      Figure 3Human Hb in the rat spleens ( a, c, e) and livers (b, d, f) 1 (a, b), 3 (c, d), and 7 (e, f) days after the bolus infusion of 20 ml/kg HbV suspension. The tissues were stained with rabbit polyclonal antibody against human Hb to examine the accumulation of human Hb from HbV. Red-stained signals correspond to human Hb, and black signals to injected carbon particles. Accumulation of human Hb is observed in both spleens and livers 1 and 3 days after infusion (a, b, c, d), while no significant accumulation is seen in both organs after 7 days (e, f). Human Hbs are located in macrophages in spleen and in Kupffer cells in liver. Scale bars, 100 μm.
      The spleen and liver accumulated the HbV particles in the macrophages and the Kupffer cells (Figure 3). A significant amount of macrophages in spleen entrapping HbV particles was seen in the red pulp zone as red-colored domains (Figure 3, a, c, and e). The total area of the red-colored portion in the red pulp zone was 31.0 ± 6.1% at one day, then it gradually decreased to 5.1 ± 2.0% after 3 days, and to less than 0.05% after 7 days (Figure 4). On the other hand, a significant amount of carbon particles was seen in the marginal zone around the white pulp, where lymph cells are located, indicating the enhanced phagocytic activity (Figure 3, a, c, and e). For the liver, Kupffer cells trapping HbV particles were seen as a red-stained area at one day after the infusion (Figure 3b). After 3 days, the HbV as well as a large amount of carbons were seen in the same position (Figure 3d). The HbV particles completely disappeared 7 days after infusion while the Kupffer cells trapping a large amount of carbon were observed which corresponded to the enhanced phagocytosis (Figure 3f). The total area of the red-colored portion was 7.6 ± 1.9% at one day, then it gradually decreased to 1.3 ± 0.2% after 3 days, and almost completely disappeared after 7 days (Figure 4).
      Figure thumbnail gr4
      Figure 4Changes in the total stained area () in the red pulp zone of spleen and in the liver after after the bolus infusion of 20 ml/kg HbV suspension. The values are mean ± SD of randomly selected five areas. *Significantly different from the value at 1 day (P < 0.005).
      There was a very slight signal with Berlin blue stain in the macrophages and the white pulp zone in the spleen and and in the Kupffer cells and Glisson's sheath in the liver at 3 and 7 days (Figure 5, a and b). However, at 14 days, no stain was confirmed either in the spleen or liver, indicating that the heme metabolism from Hb proceeded smoothly.
      Figure thumbnail gr5
      Figure 5Hemosiderin in spleen (a) and liver (b) 7 days after the bolus infusion of 20 ml/kg HbV suspension. Berlin blue stain was performed to examine the presence of hemosiderin. Slight deposition of hemosiderin is observed in both spleen (arrows in a) and liver (arrows in b). Scale bars, 100 μm. Berlin blue stain.
      The oil red O staining on all of the organs after 20 ml/kg of HbV infusion revealed that slight stains were confirmed only in liver 3 days after infusion (Figure 6). The dye, oil red O, locates in the domain of neutral lipid (e.g., triglyceride). Therefore, this result indicated that the metabolism of phospholipid components proceeded smoothly, and there was no deposition of the metabolites. In kidney and lung no significant pathological infarcts, such as capillary emboli with HbV particles, are noted in either organ (Figure 7, a and b).
      Figure thumbnail gr6
      Figure 6Lipid deposition in the liver 3 days after the bolus infusion of 20 ml/kg HbV suspension. The liver was stained with oil red O to confirm the presence of a domain of neutral lipid deposition. Slight lipid deposition is seen (arrows). Bar, 100 μm.
      Figure thumbnail gr7
      Figure 7Histology of kidney (a) and lung (b) 1 day after the bolus infusion of 20 ml/kg HbV suspension. No significant pathological changes are noted in both organs. Bars, 100 μm. HE stain.

      Histopathological Examination by Transmission Electron Microscopy

      Transmission electron microscopy (TEM) observations of the phagocytic cells clearly demonstrated the presence of HbV particles in the phagosomes and capillaries both in the liver and spleen after 1 day (Figure 8, a and b). The HbV diameter is about 250 nm which is about 1/40 of red blood cells. Since one Hb contains four irons and a resulting higher electron density, the Hb-vesicles as well as red blood cells are black in color. Some HbV are seen near the RBCs indicating that they are in capillaries and most of the HbV are captured by the phagocytic cells. After 3 days, HbV cannot be seen in the capillaries and the HbV is mostly captured by the RES. After 7 days, no phagosomes with HbV particles can be observed (Figure 8, c and d).
      Figure thumbnail gr8
      Figure 8Transmission electron microscopy of spleen (a, c) and liver (b, d), 1 (a, b) and 7 (c, d) days after infusion of HbV (20 ml/kg). The HbV particles are seen in the phagosomes (black arrows) and capillaries (white arrows) both in the spleen and liver after 1 day. After 7 days, no phagosomes with HbV particles can be observed (c, d). Bars, 5 μm.

      Blood Serum Clinical Chemistry

      The AST and ALT values before the infusion of HbV were 65.4 ± 2.7 units/l and 29. 6 ± 3.5 units/l, respectively. After the bolus infusion of HbV at 20 ml/kg dose rate, they significantly increased to 483 ± 85 units/l and 149 ± 5 units/l at 8 hours, respectively. However, after 3 days they returned to the original levels; AST was 47.5 ± 7.8 units/l and ALT was 44.0 ± 15.5 units/l. The values were almost unchanged until 14 days after the infusion.

      Discussion

      Our previous studies demonstrated the sufficient oxygen transporting capability of the HbV suspension in animal tests which was comparable with red blood cells.
      • Izumi Y
      • Sakai H
      • Hamada K
      • Takeoka S
      • Yamahata T
      • Kato R
      • Nishide H
      • Tsuchida E
      • Kobayashi K
      Physiologic responses to exchange transfusion with hemoglobin vesicles as an artificial oxygen carrier in anesthetized rats: changes in mean arterial pressure and renal cortical tissue oxygen tension.
      • Sakai H
      • Tsai AG
      • Rohlfs RJ
      • Hara H
      • Tsuchida E
      • Intaglietta M
      Microvascular responses to hemodilution with Hb vesicles as red blood cell substitutes: influence of O2 affinity.
      The clinical indications for the use of the HbV suspension as an artificial oxygen carrying fluid are estimated to be mainly preoperative or perioperative hemodilution, or resuscitation from hemorrhagic shock in emergency situations, both of which result in exchanging more than 20% of original blood with the HbV suspension. Thus the dose amount is much greater than the infusion of stealth liposomes which are clinically used as vehicles for anti-tumor drugs
      • Huang SK
      • Martin FJ
      • Jay G
      • Vogel J
      • Papahadjopoulos D
      • Friend DS
      Extravasation and transcytosis of liposomes in Kaposi's sarcoma-like dermal lesions of transgenic mice bearing the HIV tat gene.
      • Bucana CD
      • Hoyer LC
      • Schroit AJ
      • Kleinerman E
      • Fidler IJ
      Ultrastructural studies of the interaction between liposome-activated human blood monocytes and allogenic tumor cells in vitro..
      and DNA transfection.
      • Caplen NJ
      • Alton EW
      • Middleton PG
      • Dorin JR
      • Stevenson BJ
      • Gao X
      • Durham SR
      • Jeffery PK
      • Hodson ME
      • Coutelle C
      Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis.
      After circulation in the blood stream, HbV particles are finally captured by RES in the same manner as the conventional phospholipid vesicles.
      • Souhami RL
      • Patel HM
      • Ryman BE
      The effect of reticuloendothelial blockade on the blood clearance and tissue distribution of liposomes.
      • Bosworth ME
      • Hunt CA
      Liposome disposition in vivo. II: Dose dependency.
      Therefore, the influence of HbV on the RES is one issue of safety evaluation that needs to be clarified to allow the use of HbV in clinical applications.
      The carbon clearance measurement showed that the systemic phagocytic activity decreased by 40% soon after the HbV infusion. Since the circulation time of HbV at the used condition is about 20 to 30 hours, the peak of the HbV accumulation is estimated to be around 1 to 2 days after the infusion, and this corresponds to the significantly decreased phagocytic activity at 8 hours and 1 day. However, it recovered 3 days after the infusion, and then it increased by 200%. The same tendency of suppression and the succeeding enhancement of phagocytic activity was reported for other oxygen carriers, i.e., Fluosol-DA
      • Hanada H
      • Kubo T
      • Ikeda M
      • Watanabe M
      Influence of Fluosol-DA 20% on reticuloendothelial systems.
      and liposomes for cancer therapy.
      • Arndt D
      • Zeisig R
      • Eue I
      • Sternberg B
      • Fichtner I
      Antineoplastic activity of sterically stabilized alkylphosphocholine liposomes in human breast carcinomas.
      The influence of the transient decrease in phagocytic activity after the infusion of HbV should be investigated in detail, especially in the septic shock model when the defense system in a body is significantly depressed.
      • Zheng S
      • Beissinger R
      • Sherwood RL
      • McCormick DL
      • Lasic DD
      • Martin FJ
      Liposome-encapsulated hemoglobin: a red blood cell substitute.
      • Whiteford M
      • Spring A
      • Rudolph A
      • Neville L
      • Abdullah F
      • Feuerstein G
      • Rabinovici R
      Effect of liposome-encapsulated hemoglobin on the development of endotoxin-induced shock in the rat.
      However, the phagocytic activity returned to the original level at 2 weeks. The results of the serum clinical chemistry (AST and ALT) indicate that the level of liver function decreased temporarily and recovered to the normal level within 3 days. Judging from these results, the change of liver was considered transient and functional. The initial body weight loss, especially the 20 ml/kg HbV group one day after the HbV administration, may be related to the decreased appetite since the animals did not fast from food and water. However, the body weight of the HbV groups returned to that of the control groups within 3 days and they grew normally.
      In our study, simultaneous splenomegaly was observed for the HbV high dose group 3 days after infusion which corresponded to the decreased plasma concentration of HbV (half life of HbV: 20∼30 hours). However, the spleen weight did not return to the control value in 2 weeks despite the fact that the phagocytic activity returned to the control value. Histopathological examination indicated that the HbV particles were metabolized within one week, and there was no infarcts in the spleen, indicating that the sustained splenomegaly is not due to the remaining non-metabolized HbV elements but due to the increased amount of phagocytic or parenchymal cells and/or red blood cells.
      The administration of lipid microspheres (IntralipidTM) did not greatly influence the phagocytic activity and spleen weight. We selected the lipid microspheres as a control injection according to some reports which showed that lipid microspheres suppress the RES function.
      • Katz S
      • Plaisier BR
      • Folkening WJ
      • Grosfeld JL
      Intralipid adversely affects reticuloendothelial bacterial clearance.
      • Marks DH
      • Patressi J
      • Chaudry IH
      Effect of pyridoxalated stabilized stroma-free hemoglobin solution on the clearance of intravascular lipid by the reticuloendothelial system.
      • Fischer GW
      • Hunter KW
      • Wilson SR
      • Mease AD
      Diminished bacterial defenses with Intralipid.
      • Sakaeda T
      • Hirano K
      O/W lipid emulsion for parenteral drug delivery III. lipophylicity necessary for incorporation in oil particles even after intravenous injection.
      The reasons for the small amount of change should be that the total concentration of solutes in a lipid emulsion (10%) is lower than that in the HbV suspension (16 wt %), and that the metabolic route of the HbV is different from that of the lipid microspheres. It was reported that the lipid emulsions were mainly consumed by muscle and fat, with a negligible contribution from the liver and spleen.
      • Fraser I
      • Pearson H
      • Bowry V
      • Bell PRF
      The intravenous Intralipid tolerance test.
      • Rossner S
      Studies on an intravenous fat tolerance test: methodological, experimental and clinical experiences with Intralipid.
      The histopathological examination clearly demonstrated that the HbV accumulated in the liver was localized in the Kupffer cells. Hepatocytes appeared normal and lobular architecture was not affected by the HbV administration. This accumulation subsided gradually during the experimental period. The HbV accumulated in spleen was localized in macrophages in the red pulp and marginal zone. Uptake of HbV in the spleen may occur using a filtration mechanism
      • Litzinger DC
      • Buiting AMJ
      • van Rooijen N
      • Huang L
      Effect of liposome size on the circulation time and intraorgan distribution of amphipatic poly(ethylene glycol)-containing liposomes.
      and opsonization.
      • Ishiwata H
      • Sato SB
      • Kobayashi S
      • Oku M
      • Doi-Vertut A
      • Miyajima K
      Poly(ethyleneglycol) derivative of cholesterol reduces binding step of liposome uptake by murine macrophage-like cell line J774 and human hepatoma cell line HepG2.
      There was no aggregation of HbV particles in renal and pulmonary capillaries at any time. Rudolph et al
      • Rudolph AS
      • Spielberg H
      • Sparogo BJ
      • Kossovsky N
      Histopathological study following administration of liposome-encapsulated hemoglobin in the normovolemic rat.
      indicated small pulmonary and glomerular infarcts due to the trapping of large clusters of aggregated particles of the liposome-encapsulated Hb. The results of this study reflect the differences in the physicochemical characteristics of the vesicles. The particle diameter of our HbV is well-regulated to about 250 nm by the extrusion method, and the vesicular surface is modified with PEG to suppress the intervesicular aggregation
      • Yoshioka H
      Surface modification of hemoglobin-containing liposomes with polyethylene glycol prevents liposome aggregation in blood plasma.
      and this may effectively prevent embolization of capillaries and venules in microcirculation.
      • Sakai H
      • Takeoka S
      • Park SI
      • Kose T
      • Izumi Y
      • Yoshizu A
      • Nishide H
      • Kobayashi K
      • Tsuchida E
      Surface-modification of hemoglobin vesicles with polyethyleneglycol and effects on aggregation, viscosity, and blood flow during 90%-exchange transfusion in anesthetized rats.
      • Sakai H
      • Tsai AG
      • Kerger H
      • Park SI
      • Takeoka S
      • Nishide H
      • Tsuchida E
      • Intaglietta M
      Subcutaneous microvascular responses to hemodilution with red cell substitutes consisting of polyethyleneglycol-modified vesicles encapsulating hemoglobin.
      Transmission electron microscopy (TEM) one day after infusion revealed that the HbV particles were captured in the macrophages in the spleen and Kupffer cells by endocytosis and not by fusion because the intact HbV particles were present in the phagosomes. The presence of HbV near the RBC indicates the circulating HbV in vasculature. The vesicular structure of HbV completely disappeared at 7 days indicating that they are totally destroyed in the phagosomes. TEM was a very effective tool for detecting the HbV particles in tissues. The conventional method to detect liposomes (phospholipid vesicles) required encapsulation of colloid gold as a microscopic marker.
      • Huang SK
      • Lee KD
      • Hong K
      • Friend DS
      • Papahadjopoulos D
      Microscopic localization of sterically stabilized liposomes in colon carcinoma-bearing mice.
      • Rosenecker J
      • Zhang W
      • Hong K
      • Lausier J
      • Geppetti P
      • Yoshihara S
      • Papahadjopoulos D
      • Nadel JA
      Increased liposome extravasation in selected tissues: effect of substance P.
      On the other hand, the high electron density of HbV due to the highly concentrated Hb solution in the inner aqueous phase of HbV as well as in RBC provided sufficient contrast of the particle and clearer images than those displayed in other reports to our knowledge.
      Anti-human Hb antibody staining was very effective for staining the human Hb-based oxygen carriers in animal tests. The antibody recognizes the globin chain of human Hb in HbV and there is no cross-reaction with rat Hb. The stained portion mostly disappeared within 7 days both in the spleen and liver. The released heme from Hb in HbV may probably be metabolized by the inducible form of heme oxygenase-1 in the Kupffer cells in the liver
      • Goda N
      • Suzuki K
      • Naito S
      • Takeoka S
      • Tsuchida E
      • Ishimura Y
      • Tamatani T
      • Suematsu M
      Distribution of heme oxygenase isoform in rat liver: topographic basis for carbon monoxide-mediated microvascular relaxation.
      and in the spleen.
      • Braggins PE
      • Trakshel GM
      • Kutty RK
      • Maines MD
      Characterization of two heme oxygenase isoforms in rat spleen: comparison with the hematin-induced and constitutive isoforms of the liver.
      Normally, iron from a heme is stored in the ferritin molecule.
      • Finch CA
      • Huebers H
      Perspectives in iron metabolism.
      This protein has 24 subunits and encloses as many as 4500 iron atoms in the form of an aggregate of ferric hydroxide. Ferritin in the lysosomal membrane may form paracrystalline structures and eventually aggregate in mass with an iron content as high as 50%. These are hemosiderins composed of degraded protein and coalesced iron. It was reported that polymerized Hb were captured by the Kupffer cells and then hemosiderin was confirmed, and the renal tubules showed siderosis.
      • Lenz G
      • Junger H
      • Schneider M
      • Kothe N
      • Lissner R
      • Prince AM
      Elimination of pyridoxalated polyhemoglobin after partial exchange transfusion in chimpanzees.
      On the other hand, in our HbV study, there were slight Berlin blue stains only in spleen and liver after 3 and 7 days, and they completely disappeared after 14 days. As the anti-human Hb antibody staining disappeared, trace staining of hemosiderin was recognized and it completely disappeared after 14 days. This indicates that the amount of the stored hemosiderin was minimal, thus the metabolism of heme and recycling of iron molecule are suggested to be on the physiological pathway.
      As for the membrane components of HbV, it was reported that the infused lipid components of liposomes are entrapped in the Kupffer cells, and diacylphosphatidylcholine is metabolized and reused as a component of the cell membrane, or excreted in bile, especially as fatty acids’ and in exhaled air.
      • Verkade HJ
      • Derksen JT
      • Gerding A
      • Scherphof GL
      • Vonk RJ
      • Kuipers F
      Differential hepatic processing and biliary secretion of head-group and acyl chains of liposomal phosphatidylcholines.
      • Dijkstra J
      • van Galen M
      • Regts D
      • Scherphof G
      Uptake and processing of liposomal phospholipids by Kuppfer cells in vitro..
      • Matsushita Y
      • Eshima K
      • Shindo T
      • Yamamoto Y
      • Hasegawa E
      • Nishide H
      • Tsuchida E
      Clearance of tissue distribution of functionalized polymeric liposomes from the blood stream of rats.
      Liposomal cholesterol reappears in blood as lipoprotein cholesterol after entrapment in the Kupffer cells, and then is excreted in bile after entrapment of the corresponding lipoprotein by the parenchymal hepatocytes.
      • Kuipers F
      • Spanjer HH
      • Havinga R
      • Scherphof GL
      • Vonk RJ
      Lipoproteins and liposomes as in vivo cholesterol vehicles in the rat: preferential use of cholesterol carried by small unilamellar liposomes for the formation of muricholic acids.
      Even though the amount of the infused HbV is much greater than in these studies, the main vesicular components of HbV, the phospholipids and cholesterol, would gradually be metabolized in the same manner. There were small amounts of trace staining with oil red O only in liver 3 days after infusion. This indicated that the deposition of neutral lipid components such as glycerides, which may be produced during the metabolism, was minimal. However, further study is necessary to clarify the influence of a large dose of HbV, especially on a lipemic model.
      The long-term stability of HbV during storage has been a major issue for the practical use of oxygen carriers especially for emergency situations. In the previous reports the polymerization of phospholipids bearing dienoyl groups in the bilayer membrane of HbV has shown the enormous stabilization of HbV against long term storage in a freezer or as a freeze-dried powder.
      • Hosoi F
      • Omichi H
      • Akama K
      • Awai K
      • Endo S
      • Nakano Y
      Radiation-induced polymerization of phospholipid mixtures for the synthesis of artificial red blood cells.
      • Satoh T
      • Kobayashi K
      • Sekiguchi S
      • Tsuchida E
      Characteristics of artificial red cells: hemoglobin encapsulated in poly-lipid vesicles.
      • Sakai H
      • Takeoka S
      • Yokohama H
      • Nishide H
      • Tsuchida E
      Encapsulation of Hb into unsaturated lipid vesicles and γ-ray polymerization.
      However, the polymerized phospholipid is difficult to decompose or metabolize and the bilayer structure remained even after 30 days with showing cytotoxicity.
      • Akama K
      • Awai K
      • Yano Y
      • Tokuyama S
      • Nakano Y
      In vitro and in vivo stability of polymerized mixed-liposomes composed of 2,4-octadecadienoyl groups of phospholipids.
      After infusing polymerized phospholipid, the phagocytic activity remained suppressed even after 3 days, and the recovery was observed after 7 days.
      • Morizawa K
      • Akama K
      • Awai K
      • Tokuyama S
      • Satoh T
      Influence of polymerizable phospholipid vesicels encapsulated hemoglobin on the murine immune system.
      On the other hand, in our method, the storage stability of our HbV is achieved by the surface modification with PEG chains and deoxygenation.
      • Sakai H
      • Tomiyama K
      • Sou K
      • Takeoka S
      • Tsuchida E
      Polyethyleneglycol-conjugation and deoxygenation enable long-term preservation of hemoglobin-vesicles as oxygen carriers in a liquid state.
      Accordingly, our PEG-modified HbV shows rapid metabolism once trapped by RES. The PEG-lipid is susceptible to hydrolysis to release PEG chains during metabolism. The released PEG chains, which is known as an inert macromolecule, should be excreted in the urine through the kidneys.
      • Yamaoka T
      • Tabata Y
      • Ikada Y
      Distribution and tissue uptake of poly(ethylene glycol) with different molecular weights after intravenous administration to mice.
      Our HbV possesses a high encapsulation efficiency of Hb, so that the weight ratio of Hb to the total lipid component is nearly 1.6 to 1.8. This is much higher than the previously reported liposome encapsulated Hb. The reduced amount of lipid is beneficial for reducing the burden on RES. Even though several groups have tried so called liposome encapsulated Hb without polymerized phospholipid, there are few reports on the metabolism and the fate of the components. Sakaguchi et al
      • Sakaguchi K
      • Suzuki M
      • Ogata Y
      • Suzuki K
      • Kamitani T
      Biodistribution of the neo red cell and effect on the phagocytic activity of the reticuloendothelial system.
      tested the metabolism of their product, Neo Red Cells, histopathologically with the whole body autoradiograms. However, the concentration of Hb and infusion amount were lower than our case. Therefore, our study demonstrates more detailed information with a larger dose rate of HbV for a longer period of observation.
      In conclusion, the components of the infused HbV (20 ml/kg i.v.) entrapped in the RES were smoothly metabolized within one week. Even though the HbV infusion modified the phagocytic activity for two weeks, it does not seem to cause any irreversible damage to the phagocytic organs. Our results are demonstrated only in healthy rats, while rats in hemorrhagic shock or septic shock, or with an inflammation reaction, may react differently in these pathological situations. The infused amount in this study is equal to about 1.4 l in a 70-kg man and larger volume is needed for the further evaluation. Hence safety concerns cannot be totally eliminated by this study. However, our results offer important information for evaluating the safety issues of HbV for clinical use.

      Acknowledgements

      We acknowledge Dr. M. Watanabe and Dr. A. Iwamaru (Department of Surgery, Keio University School of Medicine) and Dr. M. Suematsu (Department of Biochemistry, Keio University School of Medicine) for discussing the surgical and experimental procedure, Mr. H. Abe, Ms. T. Yamauchi, and Mr. S. Kusakari (Department of Pathology, Keio University School of Medicine) for their excellent histopathological techniques, Dr. M. Takaori (Okayama Red Cross Blood Center) for discussing the phagocytic activity, Mr. H. Hara (Waseda University) for his technical assistance, and PCL Japan Company for suggestions on the tissue specimen preparations.

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