Hemoglobin-Vesicles as Oxygen Carriers
2001; Elsevier BV; Volume: 159; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)61783-x
ISSN1525-2191
AutoresHiromi Sakai, Hirohisa Horinouchi, Ken-ichi Tomiyama, Eiji Ikeda, Shinji Takeoka, Koichi Kobayashi, Eishun Tsuchida,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoHemoglobin-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. 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.1Tsuchida E Blood Substitutes: Present and Future Perspectives. Elsevier Science, Amsterdam1998Google Scholar, 2Chang TMS Blood Substitutes: Principles, Methods, Products, and Clinical Trials. 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288: 665-670PubMed Google Scholar 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,25Arndt D Zeisig R Eue I Sternberg B Fichtner I Antineoplastic activity of sterically stabilized alkylphosphocholine liposomes in human breast carcinomas.Breast Cancer Res Treat. 1997; 43: 237-246Crossref PubMed Scopus (23) Google Scholar, 28Hanada H Kubo T Ikeda M Watanabe M Influence of Fluosol-DA 20% on reticuloendothelial systems.Med Pharmacol (Igaku To Yakugaku) (in Japanese). 1982; 7: 1763-1774Google Scholar, 39van Etten EWM ten Kate MT Snijders SV Bakker-Woudenberg IAJM Administration of liposomal agents and blood clearance capacity of the mononuclear phagocyte system.Antimicrobial Agents Chemother. 1998; 42: 1677-1681PubMed Google Scholar and also its metabolism and the influence on the tissue parenchymal cells was confirmed by histopathological examination. 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.12Sakai 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.Am J Physiol Heart Circ Physiol. 2000; 279: 908-915Google Scholar, 32Takeoka S Ohgushi T Terase K Ohmori T Tsuchida E Layer-controlled hemoglobin vesicles by interaction of hemoglobin with a phospholipid assembly.Langmuir. 1996; 12: 1755-1759Crossref Scopus (71) Google Scholar, 35Sakai 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.Bioconjug Chem. 1997; 8: 15-22Crossref PubMed Scopus (130) Google Scholar 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. 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. 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/(t2−t1) × 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. 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). 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). 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. 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. 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. 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. 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. 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). 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. 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
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