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  • V. ORAL PRESENTATIONS

  • Artificial Cells, Blood Substitutes, and Biotechnology, 36: 211237, 2008ISSN: 1073-1199 print / 1532-4184 onlineDOI: 10.1080/10731190802123863

    Oral Presentations

    SIII-3Design of Second-Generation PEGylated Intramolecularly CrosslinkedHbs to Manipulate O2 Carrying CapacitySeetharama A Acharya1,2, Tao, Hu1, Dongxia Li1, Fanatao Meng1, Amy GTsai3 and Marcos Intaglietta31Departments of Physiology and Biophysics, and Medicine 2, Albert EinsteinCollege of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA, and3Department of Bioengineering, University of California San Diego, La Jolla,CA 92093, USA; [email protected]

    Limitations in formulating of hexaPEGylated uncross-linked Hbs as bloodsubstitutes are (i) very high O2 affinity independent of the chemistry of con-jugation, and (ii) weakening of interdimeric interactions of Hb in a conjuga-tion chemistry specific fashion. The generation of PEGylated intramolecularlycross-linked Hbs overcomes these disadvantages and is helpful to (i) decreasethe colloidal osmotic pressure (COP) of PEGylated Hbs; (ii) modulate the O2affinity of the PEGylated Hbs to desired levels; (iii) to achieve higher loadsof PEGylated Hbs in circulation, if needed, without the danger of kidney fil-tration. TetraPEGylation of -fumaryl Hb, a low O2 affinity central cavityintramolecularly crosslinked Hb, using extension arm facilitated (EAF) PEGy-lation and PEGylation protocols based on reductive alkylation, acylation andthiocarbamoylation chemistry have been optimized to generate a spectrum ofPEGylated Hbs with O2 affinities in the range of 12 to 35 mm Hg, at pH 7.4 andwith a cooperativity around 2.0. The reversible protection of Cys-93() of Hbduring the EAF-PEGylation has also been optimized to facilitate the manipula-tion of the O2 affinity of the product. PEGylated Hbs are significantly protectedfrom autoxidation in plasma; and unmodified Cys-93() in the final formula-tion of the PEGylated products gives a higher resistance to autoxidation. Thesenew design strategies for development of second-generation PEGylated Hbsare expected to remedy remaining potential toxicities of using acellular Hband simultaneously provide improved intravascular persistence, and facilitatecustomizing the PEGylated products for any defined clinical settings.

    SVI-1Evaluation of New Perfluorocarbon Based Oxygen Carriers in a Rat Modelof Normovolemic Hemodilution: Effect of Emulsion Viscosity osmolarityand Perfluorocarbon Concentration

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    AC Silva1, T Gardeazabal1, M Cabrera1, F Vega1,2, CI Castro1, E Gutierrez2,JC Briceno11Blood Substitutes Laboratory, Fundacion Cardioinfantil and U. of Los An-des; 2School of Veterinary Medicine, U. of La Salle, Bogota DC, [email protected]

    The objective of this project was to evaluate the efficacy and safety of new per-fluorocarbon based oxygen carriers (PFCOCs) in a rat model of hemodilution.The experiment consisted of a two-exchange normovolemic hemodilution of40% of volemia in Wistar rats. First exchange was performed with 10%-HES.Second exchange was performed with 80% PFCOC and 20% 10%-HES. FiO2was raised to 1.0 after the second exchange. In this study formulations wereevaluated according to a factorial experimental design with the following fac-tors and levels: PFC concentration (20 and 30% v/v), viscosity (normal andhigh) and osmolality (normal and high) with n = 24. A control group (n = 7)was hemodiluted with 10%-HES. Blood gasimetry, hematocrit and hemoglobincontent were measured at baseline and 15 min after each exchange. Results: Acomparison of oxygen extraction (DavO2) between the control group and thedifferent emulsions was performed. 30%-PFC emulsions transported signifi-cantly more oxygen that the control group (p0.05). The effect of emulsion osmolality and viscosity on DavO2 was notsignificant, although the 30%-PFC normoosmolar hiperviscous emulsion per-formed slightly better. All rats tolerated well the infusion of the emulsions. Inconclusion, the new PFCOCs developed appear to be efficacious and safe inthe animal model of normovolemic hemodilution. Further work is required tobetter assess the effect of emulsion viscosity and osmolality on oxygen carryingcapacity.

    PA-2Red Cell Hemoglobin Oxygen Affinity Modulates Microvascular Hemo-dynamics and Perfusion during Acute AnemiaP. Cabrales1, A.G. Tsai1,2, M. Intaglietta1,21La Jolla Bioengineering Institute and 2Department of Bioengineering, Uni-versity of California, San Diego, La Jolla, CA/USA; [email protected]

    Responses to exchange transfusion using red blood cells (RBCs) with modifiedhemoglobin (Hb) oxygen (O2) affinity were studied in the hamster windowchamber model during acute anemia to determine its role on microvascularperfusion and tissue oxygenation. Allosteric effectors were introduced in theRBCs by electroporation. Inositol hexaphosphate (IHP) and 5-hydroxymethyl-2-furfural (5HMF) were used to decrease and increase Hb-O2 affinity. In vitroP50s (partial pressure of O2 at 50% Hb saturation) were modified to 10, 25,45 and 50 mmHg, normal P50 is 32 mmHg. Allosteric effectors also decreased

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    the Hill coefficient. Anemic condition was induced by two isovolemic hemodi-lution exchanges using 6% dextran 70 kDa to 18% hematocrit (Hct). ModifiedRBCs (18% Hct in 5% albumin solution) were infused during a third exchangetransfusion. Systemic parameters, microvascular perfusion, capillary perfusion(functional capillary density, FCD) and microvascular pO2 levels were mea-sured. RBCs with P50 of 45 mmHg increased tissue pO2 and decreased O2delivery (DO2) and extraction (VO2) and RBCs with P50 of 60 mmHg re-duced FCD, microvascular flow, tissue pO2, DO2 and VO2. Erythrocytes withincreased Hb-O2 affinity maintained hemodynamic conditions, DO2 and de-creased tissue pO2. This study shows that in an anemic condition, maximaltissue pO2 does not correspond to maximal DO2 and VO2.

    Supported by NIH BRP R24-HL64395 and grants R01-HL62354, R01-HL62318 and R01-HL76182.

    SV-2Change of Cytokine Production in Intra-Abdominal Hemorrhage Model-Effect of Hemoglobin VesicleHirohisa Horinouchi, Noaki Aikawa, Mitsutomo Kohno, Yotaro Izumi, HiromiSakai, Keitaro So, Teruyuki Komatsu, Eishun Tsuchida and Koichi Kobayashi,Dept. of Surgery, Keio University, School of Medicine, Tokyo, JAPAN,Advanced Research Institute for Sci. & Eng., Waseda University [email protected]

    Aim: When intra-abdominal hemorrhage occurs, SIRS(systemic inflammatoryresponse syndrome) is sometimes encountered. The cause of this phenomenonis thought over production of inflammatory cytokines. Safety study was done toverify that intra-abdominal HbV didnt change cytokine production. Method:30% hemorrhage was induced in male Wister rats (340 g+/10 g) via rightcarotid artery. Five minutes after hemorrhage, intra-abdominal administrationof either autologous shed blood or Hemoglobin Vesicle (HbV dispersed insaline) solution was done. 30 minutes after hemorrhage, Twice volume ofsaline was used for resuscitation. After 30, 60, 90, and 180 minutes had passed,0.5 ml of blood was withdrawn through arterial line and replaced with the samevolume of saline. Serum was separated and stored in deep freezer until use.Cytokines (IL-1 alpha, beta, IL-2, -4, -6, -10, GM-CSF, Interferon gamma,TNF-alpha) were simultaneously measured using Bio-Rad Bioplex suspensionarray system. Results: After hemorrhage, shock was confirmed in each animal(40.6+/7.2 Torr). Intra-abdominal injection doesnt change MAP and HR.After resuscitation, MAP recovered to the pretreatment level and graduallydeclined. Cytokine production didnt change significantly. However, in ASBgroup, increase of TNF alpha was seen while in HbV group, there seemedno response (P=0.11). Discussion: Intrapritoneal HbV itself doesnt changeneither the cours of hemorrhagic shock nor the cytokine production duringshock. Shock with organ damage model should be further studied.

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    SV-3Resuscitation Effect and Long Term Effect of Hb Vesicle on Organ Func-tion in Beagle DogTatsuhikko Ikeda, Hirohisa Horinouchi, Mitsutomo Kohno, Yotaro Izumi,Masazumi Watanabe, Hiromi Sakai, Keitarou Sou, Eishun Tsuchida, KouichiKobayashiDepartment of Surgery, Keio University, Tokyo, Japan; Advanced ResearchInstitute for Science and Engineering, Waseda University, Tokyo, [email protected]

    Long-term safety study was carried out in 40% hemorrhage shock- resuscitatemodel in Beagle dog. (Method) Sixteen Beagle dogs were used. After stabi-lization, 40% of blood volume was bled at a rate of 20 ml/min. We kept MAPbelow 50mmHg for 1 hour. After 1 hour hemorrhagic shock, either autologousshed blood (ASB) or HbV/5%HSA solution was used for resuscitation. MAP,HR, RR, intracranial tissue hemoglobin saturation was monitored during acutephase. 4 Hours after resuscitation, dogs were extubated and were returned tothe cages. One, 3, 7, 14, 28, 56, 84, 168, 360 days after, body weight, CBC,serum chemistry was analyzed. Several dogs were sacrificed on day 28, 168,360 and histological findings were studied. (Results) Efficacy of HbV for theresuscitation of hemorrhagic shock seemed similar to that of ASB. RBC counthad recovered within 7 days after shock. HbV in blood stream decreased itsconcentration and was not detected in the blood taken on day7. After resusci-tation, AST/ALT increased twice to the normal value at day 1 in both HbV andASB group. This upregulation normalized on day three. There was no deathin both HbV and ASB group. All dogs didnt show any abnormal findingsduring experimental period. Histological exam showed no substantial changes.(Discussion) HbV is thought safe and promising blood substitute in long termstudy. Early rise of AST/ALT after resuscitation is thought to be a reaction tohypovolemic shock.

    SI-3Efficacy of Liposomal Hemoglobin (Lhb) in Massively HemorrhagedCynomolgus MonkeysT. Ishizuka, H. Goto, Y. Ogata, S. Kaneda, H. KasukawaTerumo Corp., Japan; Takanobu [email protected]

    We have developed a liposomal hemoglobin (LHb) preparation as a red bloodcells substitute and confirmed the efficacy of LHb in hemorrhaged rats anddogs but not in primates. The purpose of this study was to examine the effectsof LHb in massively hemorrhaged cynomolgus monkeys.

    Anesthetized animals received an isovolemic exchange transfusion with acolloidal solution (HespanderTM) until the hemoglobin concentration reachedabout 3 g/dL. Immediately after that, five animals each were given 20 mL/kg of

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    LHb or saline. Survival time, blood pressure, electrocardiogram (ECG), arterialblood gases and plasma lactate level were monitored during 12 hours.

    The survival rates were 4/5 in the LHb group and 2/5 in the Saline group.In the animals that survived, arterial blood pressure was maintained duringthe observation period at a level similar to the baseline in the LHb group butnot in the Saline group. Ischemic changes in ECG and elevation in plasmalactate level were observed at 12 hours in the Saline group but not in the LHbgroup. No apparent signs of pulmonary dysfunction were observed in eithergroup.

    We confirmed the efficacy of LHb as a red blood cells substitute in mas-sively hemorrhaged cynomolgus monkeys. These results suggest that LHb isalso useful in primates.

    SVII-5Administration of Hemoglobin Vesicle under Mechanical Ventilation DoesNot Affect Lung FunctionIzumi Y1, Yamada T2, Ogawa EN2, Morisaki H2, Sakai H3, Horinouchi H1,Takeda J2, Tsuchida E3, Kobayashi K11Division of General Thoracic Surgery, and 2Department of Anesthesia, Schoolof Medicine Keio University, Tokyo, Japan. 3Advanced Research Institute forSci and Eng, Waseda University, Tokyo Japan; [email protected]

    Hemoglobin Vesicle (HbV) contains concentrated hemoglobin solution withina phospholipid vesicle. As a resuscitative fluid, it will potentially be admin-istered to patients receiving care in the intensive care unit, which includesmechanical ventilation. HbV did not affect lung function when administeredunder spontaneous breathing. In this study, we administered HbV when thelung was mechanically ventilated. Rabbits were mechanically ventilated (tidalvolume, 30ml/kg), and 30% exchange transfusion was done with HbV or saline.Hemodynamics, and blood gas parameters were monitored for 4 hours. Thelung was then resected for histology. Wet to dry ratio was also measured. Therewas no apparent change in systemic blood pressure in either group. Arterialoxygen tension tended to decrease gradually, but there was no significant dif-ference between groups. Slight lung edema was observed in both groups. Therewas no significant difference in the wet to dry ration between the groups. Themoderate decrease in arterial oxygen tension suggested the presence of ventila-tion induced lung injury, but there was nothing to suggest that it was aggravatedby HbV administration. (Supported by NHLW of Japan)

    SI-2Clinical Evaluation of HemospanR As an Oxygen-Carrying Plasma Ex-pander in Surgical PatientsPE Keipert, N Winslow, RM WinslowSangart Inc., 6175 Lusk Blvd., San Diego, CA; [email protected]

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    HemospanR is a hemoglobin-based oxygen carrier (HBOC) consisting of chem-ically modified human hemoglobin (MalPEG-Hb) in Lactated Ringers solu-tion. Hemospan is formulated at low Hb concentration (4.3 g/dL), high oxygenaffinity (P50 5 mmHg) and high colloid osmotic pressure (COP 50 mmHg),properties that prevent oxygen-induced vasoconstriction while preserving cap-illary perfusion and oxygen delivery.

    Clinical studies with Hemospan in Europe and in the US have enrolled atotal of >300 subjects as of the end of July 2007. The first Phase I study inhealthy volunteers did not show any hypertension or gastrointestinal side ef-fects, which have been reported with other first-generation HBOCs. A recentlycompleted dose escalation Phase Ib/II study in orthopedic surgery patients alsoreported no serious adverse events (SAEs) at Hemospan doses ranging from200 to 1000 mL. A similar Phase II safety study in prostatectomy patients iscurrently ongoing in the US, with no safety concerns noted.

    A randomized, double-blind Phase II study of Hemospan was completed at6 Swedish hospitals in 90 patients (ASA Class I-III; age 50 to 89) undergoing hipreplacement or fracture surgery with spinal anesthesia. The primary endpointswere the incidence of hypotension and the use of vasopressors. The percentageof patients with hypotensive episodes was significantly lower (48% in the 250-mL dose group, 43% in the 500-mL dose group) compared to 84% in controls(P < 0.025). The incidence of vasopressor use was also reduced in the 250-mL(17%) and 500-mL group (13%) compared to controls (32%). Mean heart ratewas less in both treated groups versus controls (P

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    therapy in this patient population was tested. In a double blinded phase IIpilot trial, 45 patients with acute non-ST segment elevation myocardial in-farct scheduled for PCI procedure were randomized to receive IV infusionof either a colloid control or 15g or 30 g of HBOC-201. Systemic and coro-nary hemodynamic parameters were assessed at baseline, post-HBOC infu-sion and post-PCI. After HBOC-201 administration, systemic arterial bloodpressure (SBP), pulmonary capillary wedge pressure and systemic vascularresistance were elevated indicating systemic vasoconstriction. Cardiac output(CO) and mixed venous oxygen saturation (SVO2) were moderately depressed.However, the elevated SBP could be managed with traditional antihyperten-sive medications. Despite the systemic effects, coronary hemodynamic andfunctional parameters (e.g., average peak velocity, coronary blood flow, leftventricular work index) were not altered under resting and adenosine-inducedhyperemia indicating that HBOC-201 does not interfere with coronary autoreg-ulatory mechanisms. In addition, there was no angiographic (QCA) evidence ofcoronary vasoconstriction in major epicardial vessels examined. These resultsindicate that HBOC-201 could be safely administered to patients undergoingpercutaneous coronary revascularization for treatment of an acute ischemicsyndrome.

    SIII-4Three-Dimensional (3D) Solution Structure of Maleimide-Poly(ethylene)Conjugated Hemoglobin by Small-angle X-ray Scattering: Implicationsfor HemospanR as a New Oxygen TherapeuticKD Vandegriff1, A Malavalli1, DA Baker1, DI Svergun2, F Ekstrom3, CNilsson3, RM Winslow11Sangart, Inc., 6175 Lusk Blvd., San Diego, CA USA 92121; 2European Molec-ular Biology Laboratory, Hamburg Outstation, Hamburg, Germany; 3SwedishDefense Research Agency (FOI), CBRN Defense and Security, Umea, Sweden;[email protected]

    Several general benefits have been attributed to poly(ethylene) glycol(PEG) conjugation to proteins, including increased intravascular retentiontime. HemospanR is human hemoglobin conjugated to 7 linear strandsof poly(ethylene) glycol (PEG), 5-kD each. The design strategy forHemospan was initiated using poly(ethylene) glycol chemistry to increasehemoglobin macromolecular size without polymerization. Solutions of unmod-ified hemoglobin (Hb) and PEGylated hemoglobins with either two (P5K2) or7 (P5K7, Hemospan) 5-kD PEGs were evaluated using small-angle X-rayscattering (SAXS). The 3D solution structures reveal that PEGylation elon-gates the dimensions of the hemoglobin molecule: Hb < P5K2 < P5K7, givingmaximal molecular dimensions of 7, 11, and 13 nm, respectively. The overalltertiary structure of hemoglobin remains intact upon conjugation to the PEGchains, but its quaternary structure appears compacted, presumably due to the

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    dehydration of the intersubunit interfaces. The major part of the PEG chainsprotrudes away from hemoglobin, while the rest interacts with the core pro-tein. Based on calculations of the PEG Flory radius, the SAXS structure ofHemospan suggests that the PEG chains exists at the transition point betweenmushroom and brush conformations. PEGylation introduces a strong inter-molecular repulsive effect that increases with the amount of conjugated PEG.These results further define the biological activity of Hemospan as an oxygentherapeutic such that: 1) PEG provides surface shielding of the hemoglobinmolecule; 2) hemoglobin tertiary structure is not altered by PEG conjugation;and 3) PEG imparts intermolecular repulsive forces, which may affect the rateor mechanism of Hemospans intravascular clearance.

    SV-4Application of Hemoglobin Vesicles to Anemia Due to Inflammatory BowelDisease in a Mouse ModelM Kohno1, H Horinouchi1, Y Izumi1, T Ikeda1, H Sakai2, E Tsuchida2, KKobayashi11Department of Surgery, Keio University School of Medicine, Tokyo Japan2Advanced Research Institute for Science and Engineering, Waseda University,Tokyo, Japan; [email protected]

    Human inflammatory bowel disease (IBD) is a chronic, relapsing and remittinginflammatory condition of unknown origin that afflicts individuals of both sexesthroughout life. The disease is clinically characterized by two phenotypesulcerative colitis and Crohns disease. Anemia affects between 30% and 70% ofpatients and has great impact on the quality of their lives. They currently receivetransfusion of red blood cells for severe anemia. The purpose of this study wasto assess the feasibility and safety of infusion of encapsulating concentratedhuman hemoglobin (Hb vesicles) for treatment of anemia due to IBD. Infusionof Hb vesicles did not exacerbate IBD as determined by a serum biomarkerand histologic evaluation compared with saline in the dextran sulfate sodiummouse colitis model. Further, disease activity index for IBD and body weightloss tended to be decreased by infusion of Hb vesicles.

    SVI-4Genetic Engineering of Heme Pocket in Human Serum Albumin: Controlof O2 Binding of Iron Protoporphyrin IxT. Komatsu,1,2 A. Nakagawa,1 E. Tsuchida11Reserach Institute for Science & Engineering, Waseda University, Tokyo 169-8555 Japan, 2PRESTO, Japan Science and Technology Agency (JST), Saitama332-0012 Japan; [email protected]

    Complexing an iron protoporphyrin IX into a genetically engineered hemepocket of recombinant human serum albumin (rHSA) generates an artificial

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    hemoprotein, which can bind O2 in much the same way as hemoglobin (Hb).1We previously demonstrated a pair of mutations that are required to enablethe prosthetic heme group to bind O2 reversibly: (i) Ile-142His, and (ii)Tyr-161Phe or Leu [I142H/Y161F (HF) or I142H/Y161L (HL)].2) Here wereport additional new mutations designed to manipulate the architecture of theheme pocket in rHSAheme complexes by specifically altering distal aminoacids. We show that introduction of a third mutation on the distal side of theheme can modulate the O2 binding equilibrium.3) The HSA(HL/L185N)hemeshowed very high O2 binding affinity (P O21/2: 1 Torr, 22C), which is 18-foldgreater than that of the original double mutant rHSA(HL)heme and veryclose to the affinities exhibited by myoglobin and the high-affinity form of Hb.Introduction of Asn at position 185 enhances O2 binding primarily by reducingthe O2 dissociation rate constant. Replacement of polar Arg-186 with Leu orPhe increased the hydrophobicity of the distal environment, yielded a complexwith reduced O2 binding affinity (P O21/2: 910 Torr, 22C), which neverthelessis almost the same as that of human red blood cells and therefore better tunedto a role in O2 transport.

    SVII-3Microcirculation Disorders Under Chronic Venous Insufficiency: Diagnos-tics and Correction with TaxifolinVI Kozlov, VP Tihonov, LI Dergatchova, GA Azizov, OA Gurova, VV BaranovDepartment of anatomy and Department of laser medicine, Peoples FriendshipUniversity of Russia; Company Diod; Company Centre for Analysis ofSubstances, Moscow; Russia [email protected]

    The purpose of the present research consisted to investigate the influencebioflavonoid Taxipholin [Dihydroqercetin], possessing antioxydative action,on a skin microcirculation at patients with chronic venous insufficiency [CVI].45 patients with CVI [CEAP stage 2 4] have been surveyed at treatmentof 0.3% gel Taxipholin in comparison with treatment of 2% gel Troxerutine[control group]. Application of gel was made on a leg and foot daily within 3weeks. Methods: The estimation of skin microcirculation in a leg and foot wasmade by means of Laser Doppler flowmetry [LDF] [device LAKK, Lazma-Co,Rassia], and also TV-computer capillaroscopy of eponichii on 1 finger foots[Computer capillaroscope, Joint Stock Co, Russia]. A systemic microcircula-tion estimated by means of biomicroscopy of bulbar conjunctval microvessels.Results: At patients CVI in 100% of cases disorders of microcirculation arerevealed. Under biomicroscopy at them increases of permeability of their walland rheologic frustration were marked both structural changes of microvessels.The degree of insufficiency of microcirculation directly depend on weight ofcurrent CVI. The leading pathogenetic mechanism in frustration of microcir-culation are: progressive increase of the phenomena of venous stagnation on amicrovessel level, local blockade of a capillary blood flow and sharp damage

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    of barrier function of microvessel wall. The most vulnerable is a nutritive partof microvascular network. Frustration of a blood flow at CVI lead to changeof myogenic activity of arterioles and precapillaries. At LDF it is expressedin reduction of fluxmotion amplitude, spectral narrowing of oscillations anddisplacement of their dominant in high-frequency area that is a result of sup-pression the vasomotion mechanism. Application of gel Taxipholin positivelyaffects dynamics of parameters of a local and systemic microcirculation atpatients CVI. After the lead treatment improvement of microcirculation anddiminish of stagnation tissue blood flow was observed: average linear speed oferythrocytes in capillaries increased with 320 54 m/c up to 358 8,1 m/c,the degree of erythrocyte aggregation and adhesions of leukocytes decreased,the parity of arteriolar and venular diameters was normalized, the perivascularzone [on 20 25%] decreased. Comparison of gel Taxipholin to gel Trox-erutin [placebo] has shown, that at them comparable vasotropic effect. At thesame time it has noted been, that reactance of microvessels [on postural test]at patients CVI it becomes authentic above [on 11%] after treatment by gelTaxipholin. Conclusion: Taxipholin possesses vasotropic action at the level ofmicrovessels and owing to antioxydant action positively influences on normal-ization of microcirculation and decrease in permeability of capillary wall atpatients with CVI.

    SII-2Engineering Hemoglobin-Based Oxygen Carriers for TissueEngineeringAndre F PalmerDepartment of Chemical and Biomolecular Engineering, The Ohio State Uni-versity, Columbus, Ohio, USA; [email protected]

    A priori knowledge of the dissolved oxygen (O2) concentration profile withinhepatic hollow fiber (HF) bioreactors is important in designing and develop-ing an effective bioartificial liver assist device (BLAD) for eventual clinicaluse. O2 transport is limiting within such HF bioreactors due to the poor sol-ubility of O2 in aqueous culturing media, long tortuous O2 diffusion pathswithin HF membranes, and high hepatocyte O2 consumption rates. We hy-pothesize that supplementing a hepatic HF bioreactors circulating mediastream with a hemoglobin-based oxygen carrier (HBOC) will improve hepa-tocyte oxygenation. Our results (experimental and theoretical) indicate HBOCsupplementation of the circulating media stream leads to marked improve-ment in oxygen delivery, and provision of a greater range of oxygen ten-sions to hepatocytes over the length of the bioreactor. Therefore, our resultsthus support the use of HBOCs for improving oxygen delivery to hepato-cytes maintained within HF bioreactors. In general, the results of this workwill be indispensable in improving oxygenation of bioreactors used for tissueengineering.

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    SVI-2Haemorheological Characterization of a Perfluorocarbon-Based OxygenCarrier (Oxygent) in Microcirculatory Scale FlowJP PeachUniversity of Vermont, Burlington, VT, USA; [email protected]

    Vascular (geometric and topographical) and intravascular (rheological) factorsinfluence the resistance to blood flow. Addition of drugs, such as Oxygent, tothe blood may alter the haemorheology and therefore affect the cardiovascularsystems ability to adequately perfuse the tissue. In the microcirculatory sys-tem, blood exhibits complex rheological properties that are governed, in part,by vessel diameter, haematocrit ratio, temperature and shear rate. The aims ofthis study were to characterize the rheological properties of Oxygent mixturesand then evaluate its cardiac work load requirements in a physiologically rel-evant numerical model. To do this, whole blood was diluted with Oxygent orphosphate buffered saline 7.2 (PBS) to produce mixtures with 25, 30, 35 or40% haematocrit and 60% plasma. A 100-m diameter glass-tube viscometerwas used to measure the pressure drop over a 32-mm long test section at variouswall shear rates (100 1,800/sec). Viscosity was then computed and a regres-sion analysis was used to develop equations which predict viscosity from theshear rate for each haematocrit ratio group. To test the effects of temperatureon pure Oxygent, its viscosity was determined in the temperature range of 32.2 41.1C with wall shear rates between 17.5 and 2,275/sec. Lower tempera-tures led to significantly (p < 0.0005) higher viscosity of Oxygent. In an effortto interpret these results in a clinically relevant framework, cardiac workloadover a 50-segment vascular network was examined. The pressure drop over thenetwork was used as a measure of cardiac workload. The boundary conditions(inlet pressure, flow rate and haematocrit) were randomly selected for each inletsegment. The pressure drop using an Oxygent-blood mixture was compared tothe pressure drop using a PBS-blood mixture having the same boundary condi-tions. These results will provide a theoretical measure of the cardiac workloadrequired to pump an Oxygent-based blood mixture compared to a PBS- basedmixture.

    SV-1Hemoglobin-Vesicles as Artificial Oxygen Carriers: Interactions with Lig-and Molecules in the Production Process and in Blood CirculationH Sakai1, A Sato1, K Sou1, H Horinouchi2, K Kobayashi2, E Tsuchida1;1Research Institute for Sci. & Eng., Waseda University, Tokyo, Japan; 2Dept. ofSurgery, School of Medicine, Keio University, Tokyo, Japan; [email protected]

    Hb-vesicles (HbV, 250 nm) are artificial O2 carriers, and the safety and efficacyas a transfusion alternative have been clarified in detail [17]. Hb binds not onlyO2 but also CO and NO very strongly. In some conditions HbCO dissociates

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    easily. The interactions of HbV with these gaseous ligands are important bothin the production process and in blood circulation. HbCO is resistant to heatingand it enables pasteurization at 60oC for 10 hrs to guarantee the utmost safetyfrom infection [8]. The purified HbCO is concentrated to ca. 40 g/dL andencapsulated with a lipid bilayer membrane without protein denaturation. Thevesicular surface is decorated with PEG. HbCO can be easily converted to HbO2by photoirradiation in an aerobic condition. Finally O2 is completely removedout and the resulting deoxy-state HbV can be stored at room temperaturefor over 2 years [9]. HbV does not induce vasoconstriction in contrast tomolecular Hbs. This difference presumably relates to the reduced uptake ofendogenous NO and CO owing to the large dimension and the cellular structureof HbV [3,4]. Recently, we tested intravenous injection of exogenous CO-bound HbV into rats, and found out that CO was released quite promptly in3 hrs with a cytoprotective effect at reperfusion. This indicates a possibilityof a new clinical application of HbV in addition to its use as a transfusionalternative.

    PA-4Accurate and Quantitative Determinatuion of the Structural Profiles ofHemoglobin Vesicle By Means of X-Ray and Light Scattering TechniquesT. Sato1, H. Sakai1, K. Sou1, O. Glatter2, E. Tsuchida1Research Institute for Science and Engineering, Waseda University, Tokyo,Japan, and 2Institute of Chemistry, University of Graz, Graz, Austria;[email protected]

    The Waseda-type Hemoglobin vesicle (HbV), developed as a cellular-type ar-tificial oxygen carrier encapsulating a solution of purified and concentratedhuman hemoglobin (35 g/dL) with a surface-modified phospholipid bilayermembrane with poly(ethylene) glycol (PEG), has been structurally character-ized by means of the latest approaches of small-angle x-ray scattering (SAXS)and dynamic light scattering (DLS). DLS data on a diluted HbV dispersionevaluated with Optimized Regularization Technique have provided an accuratequantitative estimation of the size and size distribution of HbV; we obtainedan averaged hydrodynamic diameter of 238 nm with a narrow size distribution(standard deviation of 20 nm in volume distribution).

    A thin layer-cell DLS has for the first time made it possible to observe col-lective diffusion process of a concentrated (10g/dL) HbV dispersion withoutdilution, overcoming the interference from multiple scattering and absorptioncaused by the hemoprotein. The result confirms an ergodic (fluid-like) natureand good stability of the HbV dispersion.

    The forward SAXS intensity of HbV dispersion represents the feature ofa slightly polydisperse sphere having an averaged radius of 120 nm, whereasthe high q-part reflects the internal structure of HbV, e.g., highly concen-trated Hb (>35g/dL) and lipid bilayer. SAXS experiments have also revealed a

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    uni-lamellar structure, the thickness (5.8nm) and internal electron density pro-file of the vesicle for Hb encapsulation.

    SII-4Hemotech, a Novel Free Hemoglobin-Based Red Cell Substitute with Phar-macological Properties: the Concept and Current Status of CommercialDevelopmentJ Simoni, M. Feola, G Simoni, JF Moeller, BT Mittemeyer, AP BollonHemoBioTech, Inc., Dallas, Texas, U.S.A. and Texas Tech University HealthSciences Center, Lubbock, Texas, U.S.A; [email protected]

    The worldwide need for blood substitutes is evident; however, currently testedfree hemoglobin (Hb)-based oxygen carriers have toxicity and efficacy prob-lems. These products were developed before the recognition of Hbs intrinsictoxicity; therefore, it is not surprising that the commercial development of sev-eral first-generation products has been discontinued. The problems with theseblood substitutes revolve around blood vessel constriction and the pro-oxidantand pro-inflammatory properties of heme. To diminish intrinsic toxic effects ofHb, Texas Tech University Health Sciences Center scientists have developedand patented a novel concept of pharmacologic cross-linking and formulatedan effective free Hb-based blood substitute product. This novel blood substitute,HemoTech, that was licensed to HemoBioTech, Inc. for commercial develop-ment, is composed of purified bovine Hb, cross-linked intramolecularly withopen ring adenosine 5-triphosphate (o-ATP) and intermolecularly with openring adenosine (o-adenosine), and combined with reduced glutathione (GSH).The idea behind the use of o-adenosine was to counteract the vasoconstric-tive and pro-inflammatory properties of Hb with the activation of adenosinereceptors, which would produce vasodilatation and reduce inflammatory re-actions. The concept of conjugation of Hb with GSH was to introduce moreelectronegative charges onto the surface of Hb, which would block Hbs trans-glomerular and transendothelial passage, and would make it less visible tophagocytes. In addition, GSH shields heme from reactive oxygen species andnitric oxide. The reaction with o-ATP stabilizes the Hb tetramer, but the re-action with o-adenosine allows the formation of Hb low molecular weightpolymers with uniform electronegative charge. HemoTech was subjected topreclinical testing and clinical proof of medical concept. The results of thesestudies are favorable, indicating that HemoTech has vasodilatory activity andcan reduce vasoconstriction that follows hemorrhage, has erythropoietic ac-tivity and produces no adverse nephrotoxic, neurotoxic, oxidative, inflamma-tory or apoptotic reactions. These findings indicate that in order to designa non-toxic and efficacious free Hb-based blood substitute product, pharma-cologic cross-linking of the Hb molecule is necessary. Now, HemoTech hasentered the regulatory process for commercial development in the U.S. andabroad.

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    PA-3Effect of HemospanR on Microvessel Diameter and Functional CapillaryDensity in a Transgenic Mouse Model of Sickle Cell AnemiaAG Tsai 1,2, P Cabrales1, MA Young3, RM Winslow2,3, M Intaglietta21La Jolla Bioengineering Institute, La Jolla; 2Department of Bioengineering,University of California, San Diego, and 3Sangart Inc. San Diego, CA/USA;[email protected]

    Targeting O2 delivery to anoxic regions may reduce the incidence of com-plete deoxygenation of the RBC and thus be an effective therapy in sickle celldisease. Knockout transgenic mice (n = 6) were treated with an infusion ofHemospanR , a blood substitute that targets oxygen delivery to hypoxic areas(malemide polyethylene glycol-conjugated Hb, Sangart Inc., San Diego, CA)and then subjected to a hypoxic challenge (FiO2 = 0.08). Results were com-pared to a control group which received saline (n = 5). A 10% blood volume(BV) topload was infused (BV estimated as 6% body weight). Changes ofmicrovessel diameter and functional capillary density (number of capillariesperfused per area of tissue) were studied in vivo using the dorsal skin foldwindow chamber model. Mean arterial pressure was higher after treatmentduring normoxia and hypoxia challenge as compared to the control group. Ar-terioles dilated and venules remained unchanged from baseline during hypoxiain both study groups. FCD was statistically reduced from baseline during hy-poxia; however HemospanR infused animals maintained FCD at a level thatwas statistically higher than observed with saline infusion; 60 15% and27 24%, respectively. Thus HemospanR treatment reduces the severity ofhypoxia-mediated decline in FCD. Mechanistically the higher FCD could bedue to the higher perfusion pressure in the absence of vasoconstriction. Re-duced sickling and inflammatory responses (i.e., lesser leukocyte-adhesion)are likely concomitant mechanisms which support and enhance capillaryperfusion.

    Supported by NIH BRP Grant HL064395.

    SII-3Design of A Platelet Substitute Utilising Host Fibrinogen to EnhanceHaemostatisGF Walker1, J.A. Appleby1, SM Middleton1, AH Goodall21Haemostatix Ltd, BioCity, Nottingham, UK. 2Cardiovascular Sciences, Uni-versity of Leicester, UK; [email protected]

    A novel platelet substitute is being developed as a safe replacement for platelettransfusion. The product is designed to bind host fibrinogen (Fgn) after injectionand interact preferentially with activated platelets at the site of a wound. The Fgnbinding peptide Gly-Pro-Arg-Pro (GPRP) was conjugated to human albumin

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    microparticles (MPs). FITC-labelled Fgn binding by the MPs was determinedby flow cytometry. To assess MP incorporation into a fibrin clot, GPRP-MPswere added to platelet free plasma with thrombin (0.25 U/mL) and fibrin forma-tion monitored in a Platelet Aggregation Profiler. Impedance aggregometry wasused to assess MP interaction with unactivated and ADP-activated platelets inwhole blood rendered thrombocytopenic by centrifugation. Bleeding was mea-sured in busulfan-treated thrombocytopenic rabbits dosed with GRPR- (n =6) or control-MPs (n = 6). GPRP-MPs bound 4 times more FITC-labelledFgn than control MPs (p < 0.001). After addition of thrombin (0.25 U/mL)and GPRP- or control-MPs to plasma, only GPRP-MPs were incorporated intothe fibrin clot. Impedance aggregometry showed that GPRP-MPs in platelet-depleted blood enhanced ADP (3 M)-induced aggregation compared withcontrol MPs (Area Under Curve 12.4 2.6 vs 4.0 2.2 U; p < 0.001; n =6). Without ADP there was little aggregation with either GPRP-MPs or controlMPs. (AUC 3.6 2.0 vs 1.9 0.2 U; p = 0.19). In thrombocytopenic rabbits(platelet count 5 x106/mL 8; n = 12), blood loss was reduced in rabbitsreceiving GPRP-MPs compared with control-MPs (9.3 5.5 vs 20.1 7.6mL; p = 0.02). GPRP-MPs bind Fgn and exhibit haemostatic activity in vitroand in vivo. Fibrinogen-free GPRP-MPs have potential as an effective plateletsubstitute with significant safety and production advantages.

    SV-5Blood Banking-Induced Impairment of Red Blood Cells Flow PropertiesSaul Yedgar1, Alex Koshkaryev1, Hannah Relevy2, Nogah Manny2, GregoryBarshtein11Department of Biochemistry, Hebrew University Medical School, and2Blood Bank, Hadassah University Hospital, Jerusalem, Israel; [email protected]

    Background: Blood banking procedures are associated with damage to redblood cells (RBC), which can impair their flow properties, namely their de-formability, self-aggregability, and adherence to endothelial cells (EC) of bloodvessel walls. Transfusion of such RBC might thus introduce a circulatory riskto recipients. The present study was undertaken to comprehensively explorethe effect of cold storage and -irradiation on RBC flow properties. Methods:RBC flow properties were monitored prior to and during storage, as well asbefore and after -irradiation, as a function of shear stress, using a comput-erized cell flowproperties analyzer. Results: Routine cold-storage markedlydecreased RBC deformability, and strongly elevated their self-aggregabilityand their adherence to EC, as expressed by the amount of altered RBC andthe strength of intercellular interactions. These changes were observed alreadyat the second week of the storage period. The elevation of RBC/EC interac-tion was well correlated with translocation of phosphatidylserine to the RBCsurface. -Irradiation induced an immediate, sharp increase in the number of

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    rigid cells, but did not affect RBC adherence and aggregability. Conclusions:RBC hemodynamic behavior appears to be especially sensitive to cold storage,as it is impaired long before the expiration date of blood units, as well as to -irradiation. Since impaired RBC flow properties facilitate circulatory disor-ders, the potential circulatory risk of the practiced blood banking procedureshould be taken into account when considering RBC for transfusion.

    References: Hovav et al., Transfusion 39: 277-281, 1999; Relevy et al.,Transfusion, Published online: Sep 27th, 2007.

    SII-1Peg-Conjugated Hemoglobin (HemospanR ) Liganded with CarbonMonoxide Reduces Myocardial Infarct Size Following Ischemia /Reper-fusion in RatsMark Young, Jeff Lohman, Ashok Malavalli, Kim Vandegriff, Robert WinslowSangart, Inc. San Diego, CA, USA; [email protected]

    Carbon monoxide (CO) is reported to confer cytoprotective effects in models ofhypoxia, and ischemia. However therapeutic strategies are complicated by thelack of a satisfactory delivery method for CO. Hemospan (MP4) is a polyethy-lene glycol-conjugated human hemoglobin in development as an oxygen carry-ing plasma expander. We investigated the effects of Hemospan saturated withCO (CO-MP4) in a rat model of myocardial ischemia and reperfusion, andcompared the effects with oxy-MP4 or -Hb. Lactated Ringers (LR) andpreconditioning (PC) were employed as negative and positive controls, respec-tively. Pentobarbital-anesthetized rats were mechanically ventilated (FiO2 =0.3) and subjected to 30 min of coronary ligation followed by recovery and24 hr reperfusion. Treatment (30% blood volume topload) was begun prior toligation and continued for 24 hr. Non-ischemic area (area at risk, AAR) wasdistinguished by Evans blue and infarct size (IS) was delineated with triph-enyl tetrazolium chloride. Ischemic area was uniform in all animals as AARdid not differ between groups. Infarct size (IS/AAR) was reduced (P

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    Min Dai1, Minghua Yu1, Jianqun Han1, Hongwei Li1, Jian Zhang1, Qian Liu2,Ruijuan Xiu11Institute of Microcirculation, Chinese Academy of Medical Sciences (CAMS)& Peking Union medical college (PUMC), 2Peking Union Medical CollegeHospital (PUMCH), Beijing, China; [email protected]

    Tumor hypoxia microenvironment is strongly associated with a diminishedtherapeutic response and malignant progression. So it is then not surprisingthat hypoxia has been considered an attractive target for the development ofnovel anti-cancer therapies. A number of artificial oxygen carriers are cur-rently in advanced clinical trials for their ability to replace red blood cells andto ensure adequate tissue oxygenation. However, little has been done to inves-tigate that if intravenous administration of blood substitutes were effective inincreasing the oxygenation throughout experimental tumors. To investigate theeffect of PEG-conjugated hemoglobin on tumor hypoxia microenvironment,HeLa cells were cultured and injected to the armpit of BALB/c nude mice.PEG-conjugated hemoglobin solution was intravenous administrated twice aweek, and then the animals were sacrificed after a month. HypoxyprobeTM-1(pimonidazole hydrochloride) was used the detected the Oxygen gradients intumor tissue. ELISA and immunocytochemistry methods used for the detec-tion of HIF and VEGF protein in blood and tumor tissue separately have beendetermined. The results showed that hypoxia in tumor tissue were attenuatedby PEG-conjugated hemoglobin solution. HIF and VEGF expression were de-cline compared with the control group. Taken together, our data show thatPEG-conjugated hemoglobin suppresses the HIF and VEGF expression due tothe increase of tumor tissue oxygenation.

    SIV-1The Study on a Rat Exchange Transfusion Model By Artificial Red BloodCells (Hemoglobin Vesicle)Jing Fan, Xueqiao WangTianjin Blood Center Tianjin, P.R. China; [email protected]

    Objective: In a rat exchange transfusion model,as an artificial oxygen carrier,the security and validity with the artificial red blood cells (hemoglobin vesicle,HbV) has been verified. Methods: The first, to construct a best rat exchangetransfusion model. The second, using the model to transfuse blood with theartificial red blood cells. The finally,to evaluate the capabilities of the artificialred blood cells by some methods (such as: Blood routine test, Blood gastest, Flow cytometry and Pathology), and to compare the therapeutic effectof the different product in the rat acute hemorrhagic shock and resuscitation.Results: The artificial red blood cells (HbV) has been developed to provide theoxygen-carrying ability. The Studies have shown that after approximately 70%of hemoglobin is lost from circulation, then supply the artificial red blood cells

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    suspension,the rats survive rate is 100%. The Pathology results indicate that nosignificant change in the viscera(heart, liver, kidney, lung). Conclusion: Theartificial red blood cells (HbV) are the artificial oxygen carrier with bionicsmeaning. It can be helpful the acute hemorrhagic shock rats pass the first hourafter the severe trauma, that is golden hour.Key words: artificial red blood cells; hemoglobin vesicle; a rat exchangetransfusion model

    SVII-1Effect of PEG-Conjugated Hemoglobin Solution with Chemotherapy oncharacteristics of tumor neovascularizationJianqun Han1, Min Dai1, Minghua Yu1, Hongwei Li1, Jian Zhang1, Qian Liu2Ruijuan Xiu11 Institute of Microcirculation, Chinese Academy of Medical Sciences (CAMS)& Beijing, China Peking Union Medical College (PUMC), 2Peking UnionMedical College Hospital (PUMCH) Beijing, China; [email protected]

    It is recognized that hypoxia is a major driving force behind tumor vasculariza-tion, metastatic spread and resistance to radiation and chemotherapy. Tortuos-ity, dilation, sacculation and permeability morphology changes are the notablecharacteristics of newly formed tumor vessels which conversely worsen tumortissue oxygen provision. The aim of this study was focused on the tumor mi-crovessels morphology changes when PEG-conjugated hemoglobin combinedwith cislplatin treatment. Inoculated tumor cells to the submucosa of goldenhamsters cheek pouch to establish tumor model. Computer assisted intravitalmicroscopy was used to observe and measure microvessels tortuosity and func-tional capillary density. At 5 days post-innoculation microvessel tortuosity closeto tumor mass was significantly decreased in animals received both cisplatinand PEG-Hb (0.6 g/kg) compared to animals received single agent cisplatin.However, no significant changes was seen in animals received both cisplatinand PEG-Hb (0.3 g/kg) compared to control. Similar tendency was presented intumor functional capillary density between the two groups. The improvementof newly formed vessel structure was positive corelation with tumor tissueoxygenation. Conclusion: Higher dose of PEG-conjugated hemoglobin solu-tion with chemotherapy treatment is benefit to tumor tissue oxygenation andmicrovessel normalization. Improved tumor vasculature facilitate oxygen andchemotherapic agents transport to tumor tissue. It is indicated that the evaluatedPEG-conjugated hemoglobin may be a potencial adjuvant to chemotherapy incancer.

    SVI-3Polyoxyethylene-Modified Albumin-Heme Hybrid Synthesis, Propertyand Oxygen-Binding AbilityYB Huang1, T Komatsu2, E Tsuchida2

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    1State Key Laboratory of Polymer Physics and Chemistry, Changchun Instituteof Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, JilinProvince, P.R.China. 2Advanced Research Institute for Sciences and Technolo-gies, Waseda University, Tokyo, Japan; [email protected]

    Recombinant human serum albumin (rHSA) incorporates a synthetic heme,providing an artificial hemoprotein [albumin-heme (rHSA-heme)] which hasthe potential to bind and release f under physiological conditions (pH 7.3, 37C)in the same manner as Hb.

    In order to improve the circulation persistence in blood stream, we haverecently prepared a new albumin-heme hybrid modified by polyoxyethylene(PEG) derivatives, and its structure, property and oxygen-binding ability werecarefully investigated. The maleimido-activated or succinimide-activated PEG(Mw. 2 kDa and 5 kDa; M2000, M5000, S2000 and S5000) were used forcovering the surface of rHSA-heme by covalently bond.

    By controlling the reaction conditions, 4 types of PEG -modified rHSA-heme ([rHSA] 5 wt%) with 6 PEG chains have been synthesized; numbers of thechains were determined by MALDI TOFMS analyses. The PEG (rHSA-heme)salso bind O2 reversibly dependent of the O2 partial pressure. The O2-bindingaffinities (p1/2) were 3136 Torr and the half times of the oxy-form werearound 12 hr (37C). The viscosity of the S5000 and M5000 modified PEG(rHSA-heme) solutions showed relatively high values, while the viscosity ofS2000 and M2000 modified PEG (rHSA-heme) were nearly the same as thatof the rHSA-heme solution. The circulation persistence of the heme was alsoinvestigated by 20% top-load in rats, and the half lifetime was significantlyextended to 15 hr.

    The author would like to thank very much to Dr. T. Komatsu and Prof.E. Tsuchida from Advanced Research Institute for Sciences and Technologies,Waseda University, Japan for their great contribution on this oxygen-transfusionproject.

    SIII-2Modification of Hemoglobin by Crosslinking with a Band 3 TerminusDerived Peptide as a Blood SubstituteYL Lin, KT HuangDepartment of Chemical Engineering, National Chung Cheng University, Chia-Yi, Taiwan, China; [email protected]

    Hemoglobin solution lacking 2,3-diphosphoglycerate (2,3-DPG) is not ablerelease sufficient oxygen in the circulation of human body. In this study,therefore, a peptide composed of 9 amino acids, 7 from N terminus ofhuman erythrocytic Band 3 protein (AcMEELQDD) and cysteine and glu-tamic acid added sequentially in C terminus is crosslinked to hemoglobin

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    surface serving as a dynamic allosteric effector of hemoglobin for oxygenrelease. The derivative of polyethylene glycol (PEG), Maleimide-PEG-N-hydroxysuccinimidyl (MAL-PEG-NHS) was used to crosslink hemoglobinwith peptide. The crosslinked peptide on hemoglobin is able to modulate theoxygen affinity of hemoglobin, resulting in a higher P50 value of this peptide-modified hemoglobin (peptide-PEG-Hb) in the oxygen dissociation curve ascompared to unmodified hemoglobin. Furthermore, succinimidyl propionate-PEG-succinimidyl propionate (SPA-PEG-SPA) was used to crosslink intra-and inter-hemoglobin molecules to prevent the dissociation of hemoglobintetramers. This resulting hemoglobin polymer exhibited a similar oxygen dis-sociation behavior to peptide-PEG-Hb. Taken together, we have successfullymodified hemoglobin with a conjugated oxygen affinity modulator providing anew aspect of hemoglobin-based blood substitute.

    SVII-2Effects of Different Molecular Weight of Hydroxyethyl Starch on Hemor-rhagic Shock in Rats1,2Li Tao, 2Liu Liang-ming,1Yang Chengmin 1Tianjin Concord Biotech De-velopment Research Institute 2State Key Laboratory of Trauma, Burns andCombined Injury, Department 2Research Institute of Surgery, Daping Hospital, The Third Military Me-dial University, Chongqing 400042, China [email protected], [email protected]

    Objective: To compare the resuscitation effect of 6% different molecularweight hydroxyethyl starch(HES) on hemorrhagic shock in rats. Methods:SD rats were used to make hemorrhagic shock model by 45% hemorrhagic. Ef-fects of different molecular weight HES(HES40, HES130, HES200) on meanarterial blood pressure(MAP), left intraventricular systolic pressure(LVSP), themaximal change rate of left intraventricular pressuredp/dtmax) of hemorrhagicshock rat were observed. Meanwhile, the artery blood gas, the survival timeand 24 hour survival rate were also observed in the present study. Results:Improving hemodynamic parameter effects of our HES on hemorrhagic shockrats were similar to Voluven and Haes. HES130 and HES200 prolonging thesurvival time of shocked animals was pretty better than HES40, among thethree types of molecular weight HES, HES200 had the best effect and equive-lant to Voluven and Haes. Conclusion: Three types of molecular weight HESproduced by our company have good beneficial effect on hemorrhagic shock.HES200 has a better effect.Key Words: Hemorrhagic shock; HES; Voluven; Haes

    SIII-5Porphyrin-Histidine System Recognition of CO2 and Other Anions:mimetic of hemoglobin action in physiological conditions

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    Di Li1, Wenqian Dong1, Tianjun Liu2Chinese Academy of Medical Sciences & Institute of Biomedical Engineering,Peking Union Medical College, Tianjin 300192, China; [email protected]

    Hemoglobin plays a crucial role in life, as oxygen carrier in artery and car-bon dioxide carrier in vein, with this dual carrier activity, hemoglobin movesthe life cycle in animals and human beings. As an important research topicin biomaterials, with the aim to mimetic and substitute natural system, thereare a lot of research papers focused on the oxygen carrier system all over theworld, such as human and animal hemoglobin modification, natural or syn-thetic heme-based oxygen carriers, recombinant and transgenic hemoglobin aswell as perfluorocarbon based oxygen carriers, etc. All of these reveal that oxy-gen carrier is very important. However, the action of carbon dioxide has beenignored, and less reports pay attention to the carbon dioxide, which plays analmost the same function as oxygen in life. Since the carbon dioxide accumu-lation in life will cause disorder of acid-base equilibrium in vivo, and be lethalfor the patient suffering of lung illness. Here we design and synthesize somemodel compounds with porphyrin as platform with histidine as binding point,and try to mimetic the hemoglobin acting on carbon dioxide in physiologicalconditions and other systems.

    In porphyrin chemistry,molecular recognition has been developed rapidlyand turned out to be a front topic in recent years. As a favorable host, por-phyrin can recognize multifarious ion and bioactive molecule, which couldbe used in the field of enzyme analogue, phototherapy, molecular probe andso on. Here in this paper the following content was reported : 1. Synthesisof porphyrin-histidine system: a series of porphyrin-histidine compoundswere synthesized with either free amino groups or free imidazole groups or bothas binding points. 2. Evaluation of molecular recognition ability for differentcompounds: The effect of functional groups such as amino or imidazole groupas well as ambient environmental effect on molecular recognition was studied.Results showed that molecular recognition ability correlated tightly with thecompound structure of host and guest. For the same host, different bindingability to HCO3-, CO32-, H2PO4-, HPO42-, SO42-, SO32- were detected. 3.Comparing the absorbing CO2 ability for different compounds: On the basisof pressure balance, a simple equipment was designed to detect absorption ofcarbon dioxide. Results showed that free amino group plays a predominantrole in carbon dioxide absorption, while imidazole moiety acts less in oursystem.

    Summary: the synthetic porphyrin-histidine show the selective recogni-tion ability for different anions, and its binding ability depends on both stere-ospecific blockade and chemical structure. The absorption of carbon dioxideof these system benefits from the free amino group in the porphyrin-histidinesystem.

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    SIII-1Protection of Isolated Eat Heart by Polyethylene Glycol-BovineHemoglobin SolutionX Chang, C LongFuwai Hospitai, Beijing, P.R. China; [email protected]

    The aim of the study is to investigate the effect of polyethylene glycol-bovine(PEG-bHb),which was used as an oxygen carrier in cardioplegic solution, onthe protection of isolated rat hearts. Rat hearts were harvested to transferredto langendorff circuit and went through perfusion- cardioplegia&ischemia -reperfusion. St. Thomas cardioplegic solution was compared by 3 other cardio-plegic solutions containing different concentrations of PEG-bHb. The resultsshowed that after reperfusion, cardiac function and histological change werebetter in PEG-bHb groups, myocardial ATP contant was higher and cardiactroponin I in coronary flow was lower in in PEG-bHb groups. This studydemonstrated that PEG-bHb in cardioplegic solutions can provide better my-ocardial protection during ischemia.

    SIV-2Diffusion Behavior of Hemoglobin-Loaded Nanoparticles with PorousStructure as Oxygen CarriersY Sheng, Y Yuan, CS LiuEngineering Research Center of Biomedical Materials Under Ministry of Edu-cation, East China University of Science and Technology, Shanghai, P.R. China;[email protected]

    In this study, a series of molecular weight of poly(ethylene glycol)s (PEGs)were used as probes to evaluate the porous structure of hemoglobin-loaded par-ticles (HbP) fabricated by different process conditions and diffusion behaviorsof small molecules (such as glucose, ascorbic acid and glutathione) throughHbP were investigated. The results show that HbP with higher encapsulationefficiency (80%) and desirable diameter (70-200nm) were produced throughan improved double emulsion method; different molecular weight of PEGs,coencapsulated in the internal phase with Hb, successfully detected the porousstructure of HbP, especially the pore size which it is difficult to find a popularapparatus to observe directly; meanwhile, distinct diffusion behaviors of smallmolecules, which was dominated by the pore parameter of HbP, were obtainedbased on various preparation conditions. It is significant to understand the rela-tionship between porous structure and diffusion behavior so that the preparationof hemoglobin-loaded porous nanoparticles can be controlled, monitored andreproduced with a desired diffusion profile of small molecules.

    Many diseases are caused by tissue hypoxia that due to blood circulationobstacles, which is the most serious problem in clinics. Prof. Yang studiedmany drugs, such as oxygen carrying anti-shock drugs, the treatments drugs of

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    ischemic cardiocerebrovascular diseases and so on in recent years, the reportsof preliminary studies on the pharmacodynamics is below: Our preliminaryresults showed that poly-human hemoglobin solution has capacities of oxygendelivery together with remaining red blood cells, indicating potential clinicalapplications in improving or curing microcirculation disorder. Experimentsof perfusion and preservation of isolated organs demonstrated that our prod-ucts could significantly protect hearts from ischemia/reperfusion injury andappear improving the function of rat hearts after prolonged storage. We alsostudy blood flow in Golden Hamsters microcirculation. 13 male hamsterswere randomly divided into two groups. Control group include 5 hamsters withACD anticoagulant fresh blood of health people and Experimental group in-clude 7 hamsters with human polymerized hemoglobin The whole exchangetransfusion volume was up to 40% and each mouse was completed within15 minutes. Slow blood flow, stagnation, dyspnea, systemic convulsion andfinally asystole appeared immediately in five mice of control group duringthe period of exchange transfusion. Five mice were all dead within twentyminutes. But only one mouse of the experimental group showed abnormalreactions and was dead after 30 minutes. The remaining six mices microcircu-lation kept well and all survived. In short, preliminary study demonstrates thatpoly-hemoglobin solution has potential clinical application in improving orcuring disease of microcirculation disorder and perfusion and preservation ofisolated organ.

    SIV-5Reconstruction of Erythrocyte and Its Cellular Mechanical PropertiesXiang Wang, Wei Gao, Li Yang, Wei-yan Peng, Jia-xin XieCollege of Bioengineering, Chongqing University, Chongqing P.R.China;[email protected]

    Erythrocyte shape and its biomechanical properties have close relation to itsfunction. In this research the erythrocyte was reconstructed with natural struc-ture protein and lipids based on cellular mechanics and hemorheology concepts;the biomechanical properties of the reconstructed erythrocyte were studied. Theexperimental results indicated that the reconstructed erythrocytes were similarto the natural erythrocyte: having biconcave disc shape, good deformabilityand carrying-releasing oxygen function. The study implied the physiologicalfunction of reconstructed erythrocyte were similar to that of natural erythrocyte.

    SIII-6Effect of Polymerization Reaction on Cation Exchange Chromatographyon Molecular Weight Distribution of Polymerized Hemoglobin-Based Oxy-gen CarriersX Wang, L Huang, CM Yang, JF Wang

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    Tianjin University; Tianjin, P.R. China, Institute of Tianjin Uion biotechnologydevelopment company, Tianjin, P.R. China, Tianjin Polytechnology University,Tianjin, P.R. China; [email protected], [email protected] substitutes based on glutaraldehyde cross-linked hemoglobin (PolyHb)are currently being developed for use in human subjects needing blood transfu-sions. Despite the commercial development of PolyHb dispersions, it seems thatthe glutaraldehyde molecule is unsuitable cross-linker for a number of reasons:it is a dialdehyde (too reactive), it is too long a molecule (facilitating interte-tramer cross-links), and it lacks any intrinsic or functional specificity, leadingto uncontrolled polymerization. For these reasons, a new method, polymeriza-tion reaction on cation exchange chromatography (PRCEC), is established toprovide control of cross-linking (Mw). When Hemoglobin is modified or poly-merized by glutaraldehyde, the magnitude of positive charge on the moleculereduces. Because the strength of binding depends on the size of charge car-ried on molecules in cation exchange chromatography (CEC), hemoglobin orpolyHb with low modification or polymerization is enriched. If the polymer-ization takes place in this asymmetrical system, they have the more reactionodd than hemoglobin (polyHb) with high modification (polymerization). So,polyHb with narrow molecular weight distribution and low average molecularweight may be prepared by this way. The results of this study show that prod-uct polymerized by PRCEC (Hb:GDA molar ratio1:12) is polyHb with twotetramers (128kD) to four tetramers (256kD) with the yield of about 53%.

    S1-4Dextran-HemoglobinJ. Tze-Fei WongDepartment of Biochemistry and Applied Genomics Center, Hong KongUniversity of Science & Technology, Clear Water Bay, Hong Kong, [email protected] (DxHb) consists of a covalent conjugate between humanhemoglobin and dextran MW20,000. It was the first blood substitute to allow acomplete red blood cell replacement in an exchange transfusion model followedby spontaneous recovery under room air (1). It is equally effective in thehemorrhagic shock model (2). Its function as an HBOC is conferred with thefollowing advantages:

    Ease of preparation through coupling between hemoglobin and activated dex-tran (3).

    Long circulation half life of 2.4 days, or 1.9 days after correction for methe-moglobin formation (1).

    Non-entry into either kidneys or lymph (1).Utility both as an HBOC and as an oxygen sequestering agent to enhance the

    efficiency of cancer radiation therapy (4).

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    1. Tsai, S.P. and Wong, J.T. (1997). Advances in Blood Substitutes, ed.Winslow, R.M. et al, Birkhauser. P. 233247.

    2. Wong, J.T. and Blumenstein, J. (2007). Blood Substitutes, ed. Winslow,R.R. Elsevier. P. 483487.

    3. Xue, H. and Wong, J.T. (1994). Methods Enzymol. 231: 308322.4. Hill, R.P., Porter, L.S., Ives, S.A. and Wong, J.T. (1984). Int. J. Radiation

    Oncology Biol. Phys. 10: 369373.

    PA-1Inhaled Nitric Oxide Prevents Vasoconstriction after TetramericHemoglobin InfusionBinglan Yu1, Michael J. Raher1, Rong Liu1, Kenneth D. Bloch1,2, FumitoIchinose1,2, Warren M. Zapol11Department of Anesthesia and Critical Care, 2Cardiovascular Research Cen-ter, Massachusetts General Hospital, Harvard Medical School, Boston, MA,U.S.; 02114 [email protected]

    Background: Hemoglobin (Hb)-based oxygen carriers (HBOCs) have longbeen investigated for their potential use as blood substitutes. Clinical develop-ment of HBOCs has been limited by the adverse side effect of intense vasocon-striction that is attributed to scavenging of endothelial nitric oxide (NO). Theobjective of our study was to investigate whether pretreatment with inhaledNO could prevent the systemic hypertension caused by infusion of autologoustetrameric Hb solution in mice. Methods: Tetrameric Hb solution (4 g/dl) wasprepared from whole blood of C57BL/6 mice, and administered IV (0.012 ml/gBW) following a period of breathing either air or NO gas (80 ppm). Systolicblood pressure (SBP) was measured non-invasively in awake mice by tail-cuffmethod, and in anesthetized mice by invasive hemodynamics. Results: Afteradministration of tetrameric Hb, SBP increased from 118 3 to 143 7 mmHg(p < 0.05) in mice breathing air alone. In contrast, when mice were pretreatedwith inhaled NO (80 ppm, 15 min), subsequent administration of tetramericHb while breathing air did not alter SBP (118 3 vs. 120 2 mmHg, p =NS). These findings were confirmed using invasive hemodynamic methods.Breathing NO prior to tetrameric Hb administration did not increase the metHblevel in plasma (4 1% at 10 min after administration). However, 80 ppm NObreathing continued for 10 min after tetrameric Hb administration, markedlyincreased plasma metHb levels (74 10%). Conclusions: Pretreatment ofmice with 80 ppm iNO for 15 min prevents the systemic vasoconstrictor effectsof subsequent IV tetrameric Hb infusion without causing methemoglobinemia.

    SIV-4Influence of PEG-conjugated Hemoglobin on Tumor Oxygenation andResponse to Chemotherapy

  • 236 Oral Presentations

    Minghua Yu1, Jianqun Han1, Min Dai1, Hongwei Li1, Jian Zhang1, Qian Liu2,Ruijuan Xiu11Institute of Microcirculation, Peking Union Medical College (PUMC) & Chi-nese Academy of Medical Sciences (CAMS), Beijing, China; 2Peking UnionMedical College Hospital (PUMCH), Beijing, China [email protected]

    The presence of hypoxia in solid tumors has been recognized for more than50 years. Hypoxic tumors are significantly more malignant, metastatic, radio-and chemoresistant and have a poor prognosis. a number of therapeutic strate-gies have also been established to overcome tumor hypoxia by improvingoxygen supply either by oxygen or carbogen breathing or by increasing thehemoglobin level and oxygen delivery. The use of artificial oxygen carriersrepresents a new approach to the problem of hypoxia. In the present study,Female athymic BALB/c nude mice bearing the cervical carcinoma were un-treated or treated with cisplatin (5 mg/kg i.p.) to determine whether admin-istration of PEG-conjugated hemoglobin (0.3 g/kg i.v. or 0.6 g/kg i.v.) couldimprove the tumor oxygenation and to determine whether preadministration ofPEG-conjugated hemoglobin could enhance the anti-tumor efficacy of cisplatin.Hypoxia marker pimonidazole staining was employed to detect tumor tissueoxygenation status and treatment efficacy was determined by measurements oftumor volume, tumor growth delay and the extent of tumor apoptosis. We foundthat The application of higher dose (0.6 g/kg) PEG-hemoglobin solution couldsignificantly ameliorate the hypoxic condition in cervical carcinoma xenograftmodels. Co-administration of PEG-conjugated hemoglobin (0.6 g/kg) with cis-platin produced signifficant tumor growth inhibition and a reduction in tumorvasculature as compared to cisplatin alone, while the bodyweight loss as an in-dicator of toxicity was more pronounced in the combination treatment groups.Collectively, the evaluated PEG-hemoglobin in this experiment may have pos-itive effects on cisplatin or cisplatin-based chemotherapy, further work stillneeds to be carried out to minimize its potential toxicities.

    SIV-3Reduction and Suppression of Methemoglobin Loaded In the PolymericNanoparticles Used As BloodSubstitutesXL Zhang, YY, CS LiuEngineering Research Center for Biomedical Materials of Ministry ofEducation, East China University of Science and Technology, Shanghai;[email protected]

    A novel nonenzymatic reduction and suppression route, combination a fastpre-reduction by sodium dithionite (SD) and a sustaining post-reduction byreducing agents present in human plasma, was developed to control the metHb

  • Oral Presentations 237

    in bovine Hb-loaded nanoparticles (HbP) with porous microstructure to a de-sirable level. Meanwhile, the influences of the preparation factors were alsooptimized. In the pre-reduction, the metHb in the raw Hb could be reduced fromover 90% to 1.2% using SD combination with gel filtration. In double emul-sion preparation, lower emulsion strength, PEGylated polymer and additionof miscible solvent, such as acetonitrile could pronouncedly suppress metHbformation. The resultant metHb level in HbP under the optimal conditions wasabout 5.6%, which can be further reduced to 1.4% by the reducing agents inplasma with the help of superoxide dismutatse and catalase, named as the sus-taining post-reduction. The results achieved are promising for the fabricationof blood substitutes with controlled metHb level, which can fulfill the functionof binding/delivering oxygen to tissues in vivo for future trials.