V.

ORAL PRESENTATIONS
Articial Cells, Blood Substitutes, and Biotechnology, 36: 211237, 2008
ISSN: 1073-1199 print / 1532-4184 online
DOI: 10.1080/10731190802123863
Oral Presentations
SIII-3
Design of Second-Generation PEGylated Intramolecularly Crosslinked
Hbs to Manipulate O
2
Carrying Capacity
Seetharama A Acharya
1,2
, Tao, Hu
1
, Dongxia Li
1
, Fanatao Meng
1
, Amy G
Tsai
3
and Marcos Intaglietta
3
1
Departments of Physiology and Biophysics, and Medicine
2
, Albert Einstein
College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA, and
3
Department of Bioengineering, University of California San Diego, La Jolla,
CA 92093, USA; acharya@aecom.yu.edu
Limitations in formulating of hexaPEGylated uncross-linked Hbs as blood
substitutes are (i) very high O
2
afnity independent of the chemistry of con-
jugation, and (ii) weakening of interdimeric interactions of Hb in a conjuga-
tion chemistry specic fashion. The generation of PEGylated intramolecularly
cross-linked Hbs overcomes these disadvantages and is helpful to (i) decrease
the colloidal osmotic pressure (COP) of PEGylated Hbs; (ii) modulate the O
2
afnity of the PEGylated Hbs to desired levels; (iii) to achieve higher loads
of PEGylated Hbs in circulation, if needed, without the danger of kidney l-
tration. TetraPEGylation of -fumaryl Hb, a low O
2
afnity central cavity
intramolecularly crosslinked Hb, using extension arm facilitated (EAF) PEGy-
lation and PEGylation protocols based on reductive alkylation, acylation and
thiocarbamoylation chemistry have been optimized to generate a spectrum of
PEGylated Hbs with O
2
afnities in the range of 12 to 35 mmHg, at pH7.4 and
with a cooperativity around 2.0. The reversible protection of Cys-93() of Hb
during the EAF-PEGylation has also been optimized to facilitate the manipula-
tion of the O
2
afnity of the product. PEGylated Hbs are signicantly protected
from autoxidation in plasma; and unmodied Cys-93() in the nal formula-
tion of the PEGylated products gives a higher resistance to autoxidation. These
new design strategies for development of second-generation PEGylated Hbs
are expected to remedy remaining potential toxicities of using acellular Hb
and simultaneously provide improved intravascular persistence, and facilitate
customizing the PEGylated products for any dened clinical settings.
SVI-1
Evaluation of NewPeruorocarbon Based Oxygen Carriers in a Rat Model
of Normovolemic Hemodilution: Effect of Emulsion Viscosity osmolarity
and Peruorocarbon Concentration
211
212 Oral Presentations
AC Silva
1
, T Gardeazabal
1
, M Cabrera
1
, F Vega
1,2
, CI Castro
1
, E Gutierrez
2
,
JC Brice no
1
1
Blood Substitutes Laboratory, Fundacion Cardioinfantil and U. of Los An-
des;
2
School of Veterinary Medicine, U. of La Salle, Bogota DC, Colombia.
jbriceno@uniandes.edu.co
The objective of this project was to evaluate the efcacy and safety of new per-
uorocarbon based oxygen carriers (PFCOCs) in a rat model of hemodilution.
The experiment consisted of a two-exchange normovolemic hemodilution of
40% of volemia in Wistar rats. First exchange was performed with 10%-HES.
Second exchange was performed with 80% PFCOC and 20% 10%-HES. FiO
2
was raised to 1.0 after the second exchange. In this study formulations were
evaluated according to a factorial experimental design with the following fac-
tors and levels: PFC concentration (20 and 30% v/v), viscosity (normal and
high) and osmolality (normal and high) with n = 24. A control group (n = 7)
was hemodiluted with 10%-HES. Blood gasimetry, hematocrit and hemoglobin
content were measured at baseline and 15 min after each exchange. Results: A
comparison of oxygen extraction (DavO
2
) between the control group and the
different emulsions was performed. 30%-PFC emulsions transported signi-
cantly more oxygen that the control group (p<0.01). There was no statistical
difference in DavO
2
between the 20%-PFC emulsions and the control group
(p>0.05). The effect of emulsion osmolality and viscosity on DavO
2
was not
signicant, although the 30%-PFC normoosmolar hiperviscous emulsion per-
formed slightly better. All rats tolerated well the infusion of the emulsions. In
conclusion, the new PFCOCs developed appear to be efcacious and safe in
the animal model of normovolemic hemodilution. Further work is required to
better assess the effect of emulsion viscosity and osmolality on oxygen carrying
capacity.
PA-2
Red Cell Hemoglobin Oxygen Afnity Modulates Microvascular Hemo-
dynamics and Perfusion during Acute Anemia
P. Cabrales
1
, A.G. Tsai
1,2
, M. Intaglietta
1,2
1
La Jolla Bioengineering Institute and
2
Department of Bioengineering, Uni-
versity of California, San Diego, La Jolla, CA/USA; pcabrales@ucsd.edu
Responses to exchange transfusion using red blood cells (RBCs) with modied
hemoglobin (Hb) oxygen (O
2
) afnity were studied in the hamster window
chamber model during acute anemia to determine its role on microvascular
perfusion and tissue oxygenation. Allosteric effectors were introduced in the
RBCs by electroporation. Inositol hexaphosphate (IHP) and 5-hydroxymethyl-
2-furfural (5HMF) were used to decrease and increase Hb-O
2
afnity. In vitro
P50s (partial pressure of O
2
at 50% Hb saturation) were modied to 10, 25,
45 and 50 mmHg, normal P50 is 32 mmHg. Allosteric effectors also decreased
Oral Presentations 213
the Hill coefcient. Anemic condition was induced by two isovolemic hemodi-
lution exchanges using 6% dextran 70 kDa to 18% hematocrit (Hct). Modied
RBCs (18% Hct in 5% albumin solution) were infused during a third exchange
transfusion. Systemic parameters, microvascular perfusion, capillary perfusion
(functional capillary density, FCD) and microvascular pO
2
levels were mea-
sured. RBCs with P50 of 45 mmHg increased tissue pO
2
and decreased O
2
delivery (DO
2
) and extraction (VO
2
) and RBCs with P50 of 60 mmHg re-
duced FCD, microvascular ow, tissue pO
2
, DO
2
and VO
2
. Erythrocytes with
increased Hb-O
2
afnity maintained hemodynamic conditions, DO
2
and de-
creased tissue pO
2
. This study shows that in an anemic condition, maximal
tissue pO
2
does not correspond to maximal DO
2
and VO
2
.
Supported by NIH BRP R24-HL64395 and grants R01-HL62354, R01-
HL62318 and R01-HL76182.
SV-2
Change of Cytokine Production in Intra-Abdominal Hemorrhage Model-
Effect of Hemoglobin Vesicle
Hirohisa Horinouchi, Noaki Aikawa, Mitsutomo Kohno, Yotaro Izumi, Hiromi
Sakai, 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 hori-
nouc@sc.itc.keio.ac.jp
Aim: When intra-abdominal hemorrhage occurs, SIRS(systemic inammatory
response syndrome) is sometimes encountered. The cause of this phenomenon
is thought over production of inammatory cytokines. Safety study was done to
verify that intra-abdominal HbV didnt change cytokine production. Method:
30% hemorrhage was induced in male Wister rats (340 g+/10 g) via right
carotid artery. Five minutes after hemorrhage, intra-abdominal administration
of either autologous shed blood or Hemoglobin Vesicle (HbV dispersed in
saline) solution was done. 30 minutes after hemorrhage, Twice volume of
saline 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 same
volume 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 suspension
array system. Results: After hemorrhage, shock was conrmed 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 gradually
declined. Cytokine production didnt change signicantly. However, in ASB
group, increase of TNF alpha was seen while in HbV group, there seemed
no response (P=0.11). Discussion: Intrapritoneal HbV itself doesnt change
neither the cours of hemorrhagic shock nor the cytokine production during
shock. Shock with organ damage model should be further studied.
214 Oral Presentations
SV-3
Resuscitation Effect and Long Term Effect of Hb Vesicle on Organ Func-
tion in Beagle Dog
Tatsuhikko Ikeda, Hirohisa Horinouchi, Mitsutomo Kohno, Yotaro Izumi,
Masazumi Watanabe, Hiromi Sakai, Keitarou Sou, Eishun Tsuchida, Kouichi
Kobayashi
Department of Surgery, Keio University, Tokyo, Japan; Advanced Research
Institute for Science and Engineering, Waseda University, Tokyo, Japan
tatsu@sc.itc.keio.ac.jp
Long-term safety study was carried out in 40% hemorrhage shock- resuscitate
model 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 MAP
below 50mmHg for 1 hour. After 1 hour hemorrhagic shock, either autologous
shed blood (ASB) or HbV/5%HSA solution was used for resuscitation. MAP,
HR, RR, intracranial tissue hemoglobin saturation was monitored during acute
phase. 4 Hours after resuscitation, dogs were extubated and were returned to
the cages. One, 3, 7, 14, 28, 56, 84, 168, 360 days after, body weight, CBC,
serum chemistry was analyzed. Several dogs were sacriced on day 28, 168,
360 and histological ndings were studied. (Results) Efcacy of HbV for the
resuscitation of hemorrhagic shock seemed similar to that of ASB. RBC count
had recovered within 7 days after shock. HbV in blood stream decreased its
concentration 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 and
ASB group. This upregulation normalized on day three. There was no death
in both HbV and ASB group. All dogs didnt show any abnormal ndings
during experimental period. Histological exam showed no substantial changes.
(Discussion) HbV is thought safe and promising blood substitute in long term
study. Early rise of AST/ALT after resuscitation is thought to be a reaction to
hypovolemic shock.
SI-3
Efcacy of Liposomal Hemoglobin (Lhb) in Massively Hemorrhaged
Cynomolgus Monkeys
T. Ishizuka, H. Goto, Y. Ogata, S. Kaneda, H. Kasukawa
Terumo Corp., Japan; Takanobu Ishizuka@terumo.co.jp
We have developed a liposomal hemoglobin (LHb) preparation as a red blood
cells substitute and conrmed the efcacy of LHb in hemorrhaged rats and
dogs but not in primates. The purpose of this study was to examine the effects
of LHb in massively hemorrhaged cynomolgus monkeys.
Anesthetized animals received an isovolemic exchange transfusion with a
colloidal solution (Hespander
TM
) until the hemoglobin concentration reached
about 3 g/dL. Immediately after that, ve animals each were given 20 mL/kg of
Oral Presentations 215
LHb or saline. Survival time, blood pressure, electrocardiogram(ECG), arterial
blood 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 during
the observation period at a level similar to the baseline in the LHb group but
not in the Saline group. Ischemic changes in ECG and elevation in plasma
lactate level were observed at 12 hours in the Saline group but not in the LHb
group. No apparent signs of pulmonary dysfunction were observed in either
group.
We conrmed the efcacy of LHb as a red blood cells substitute in mas-
sively hemorrhaged cynomolgus monkeys. These results suggest that LHb is
also useful in primates.
SVII-5
Administration of Hemoglobin Vesicle under Mechanical Ventilation Does
Not Affect Lung Function
Izumi Y
1
, Yamada T
2
, Ogawa EN
2
, Morisaki H
2
, Sakai H
3
, Horinouchi H
1
,
Takeda J
2
, Tsuchida E
3
, Kobayashi K
1
1
Division of General Thoracic Surgery, and
2
Department of Anesthesia, School
of Medicine Keio University, Tokyo, Japan.
3
Advanced Research Institute for
Sci and Eng, Waseda University, Tokyo Japan; yotaroizumi@yahoo.co.jp
Hemoglobin Vesicle (HbV) contains concentrated hemoglobin solution within
a phospholipid vesicle. As a resuscitative uid, it will potentially be admin-
istered to patients receiving care in the intensive care unit, which includes
mechanical ventilation. HbV did not affect lung function when administered
under spontaneous breathing. In this study, we administered HbV when the
lung was mechanically ventilated. Rabbits were mechanically ventilated (tidal
volume, 30ml/kg), and 30%exchange transfusion was done with HbVor saline.
Hemodynamics, and blood gas parameters were monitored for 4 hours. The
lung was then resected for histology. Wet to dry ratio was also measured. There
was no apparent change in systemic blood pressure in either group. Arterial
oxygen tension tended to decrease gradually, but there was no signicant dif-
ference between groups. Slight lung edema was observed in both groups. There
was no signicant difference in the wet to dry ration between the groups. The
moderate decrease in arterial oxygen tension suggested the presence of ventila-
tion induced lung injury, but there was nothing to suggest that it was aggravated
by HbV administration. (Supported by NHLW of Japan)
SI-2
Clinical Evaluation of Hemospan
R
As an Oxygen-Carrying Plasma Ex-
pander in Surgical Patients
PE Keipert, N Winslow, RM Winslow
Sangart Inc., 6175 Lusk Blvd., San Diego, CA; lbarkley@sangart.com
216 Oral Presentations
Hemospan
R
is a hemoglobin-based oxygen carrier (HBOC) consisting of chem-
ically modied human hemoglobin (MalPEG-Hb) in Lactated Ringers solu-
tion. Hemospan is formulated at low Hb concentration (4.3 g/dL), high oxygen
afnity (P50 5 mmHg) and high colloid osmotic pressure (COP50 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 a
total of >300 subjects as of the end of July 2007. The rst Phase I study in
healthy volunteers did not show any hypertension or gastrointestinal side ef-
fects, which have been reported with other rst-generation HBOCs. A recently
completed dose escalation Phase Ib/II study in orthopedic surgery patients also
reported no serious adverse events (SAEs) at Hemospan doses ranging from
200 to 1000 mL. A similar Phase II safety study in prostatectomy patients is
currently ongoing in the US, with no safety concerns noted.
Arandomized, double-blind Phase II study of Hemospan was completed at
6 Swedish hospitals in 90 patients (ASAClass I-III; age 50 to 89) undergoing hip
replacement or fracture surgery with spinal anesthesia. The primary endpoints
were the incidence of hypotension and the use of vasopressors. The percentage
of patients with hypotensive episodes was signicantly 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 rate
was less in both treated groups versus controls (P<0.02), with no differences
in PR or QTc intervals (from 24-hour Holter ECG monitoring). Three SAEs
were reported, but none was related to treatment.
Hemospan is currently being evaluated in two parallel Phase III studies as
an oxygen-carrying plasma expander for surgical patients undergoing primary
hip arthroplasty, to prevent and treat the hypotension and functional hypov-
olemia associated with spinal anesthesia and surgical bleeding. By improving
hemodynamic stability during surgery and in the early postoperative period and
providing oxygen transport, oncotic pressure and plasma volume expansion,
Hemospan treatment may also reduce the incidence of postoperative morbidity.
These two randomized, double-blind Phase III studies will enroll a total of 830
patients at approximately 36 sites in six European countries.
PA-5
Effect of Iv Hboc-201 on Coronary Hemodynamics and Function in Pa-
tients Undergoing Elective Pci
Hae Won Kim, and the COR-001 Study Group Biopure Corporation, Cam-
bridge, MA 02141, U.S.A; HWKim@Biopure.com
Hemoglobin based oxygen carriers (HBOCs) are potential therapeutic ad-
juncts to patients with acute coronary syndrome undergoing elective percuta-
neous coronary intervention (PCI). Feasibility of intravenous (IV) HBOC-201
Oral Presentations 217
therapy in this patient population was tested. In a double blinded phase II
pilot trial, 45 patients with acute non-ST segment elevation myocardial in-
farct scheduled for PCI procedure were randomized to receive IV infusion
of 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 blood
pressure (SBP), pulmonary capillary wedge pressure and systemic vascular
resistance were elevated indicating systemic vasoconstriction. Cardiac output
(CO) and mixed venous oxygen saturation (SVO
2
) were moderately depressed.
However, the elevated SBP could be managed with traditional antihyperten-
sive medications. Despite the systemic effects, coronary hemodynamic and
functional parameters (e.g., average peak velocity, coronary blood ow, left
ventricular work index) were not altered under resting and adenosine-induced
hyperemia indicating that HBOC-201 does not interfere with coronary autoreg-
ulatory mechanisms. In addition, there was no angiographic (QCA) evidence of
coronary vasoconstriction in major epicardial vessels examined. These results
indicate that HBOC-201 could be safely administered to patients undergoing
percutaneous coronary revascularization for treatment of an acute ischemic
syndrome.
SIII-4
Three-Dimensional (3D) Solution Structure of Maleimide-Poly(ethylene)
Conjugated Hemoglobin by Small-angle X-ray Scattering: Implications
for Hemospan
R
as a New Oxygen Therapeutic
KD Vandegriff
1
, A Malavalli
1
, DA Baker
1
, DI Svergun
2
, F Ekstr om
3
, C
Nilsson
3
, RM Winslow
1
1
Sangart, Inc., 6175 Lusk Blvd., San Diego, CA USA 92121;
2
European Molec-
ular Biology Laboratory, Hamburg Outstation, Hamburg, Germany;
3
Swedish
Defense Research Agency (FOI), CBRN Defense and Security, Ume a, Sweden;
lbarkley@sangart.com
Several general benets have been attributed to poly(ethylene) glycol
(PEG) conjugation to proteins, including increased intravascular retention
time. Hemospan
R
is human hemoglobin conjugated to 7 linear strands
of poly(ethylene) glycol (PEG), 5-kD each. The design strategy for
Hemospan was initiated using poly(ethylene) glycol chemistry to increase
hemoglobin macromolecular size without polymerization. Solutions of unmod-
ied hemoglobin (Hb) and PEGylated hemoglobins with either two (P5K2) or
7 (P5K7, Hemospan) 5-kD PEGs were evaluated using small-angle X-ray
scattering (SAXS). The 3D solution structures reveal that PEGylation elon-
gates the dimensions of the hemoglobin molecule: Hb <P5K2 <P5K7, giving
maximal molecular dimensions of 7, 11, and 13 nm, respectively. The overall
tertiary structure of hemoglobin remains intact upon conjugation to the PEG
chains, but its quaternary structure appears compacted, presumably due to the
218 Oral Presentations
dehydration of the intersubunit interfaces. The major part of the PEG chains
protrudes away from hemoglobin, while the rest interacts with the core pro-
tein. Based on calculations of the PEG Flory radius, the SAXS structure of
Hemospan suggests that the PEG chains exists at the transition point between
mushroom and brush conformations. PEGylation introduces a strong inter-
molecular repulsive effect that increases with the amount of conjugated PEG.
These results further dene the biological activity of Hemospan as an oxygen
therapeutic such that: 1) PEG provides surface shielding of the hemoglobin
molecule; 2) hemoglobin tertiary structure is not altered by PEG conjugation;
and 3) PEG imparts intermolecular repulsive forces, which may affect the rate
or mechanism of Hemospans intravascular clearance.
SV-4
Application of Hemoglobin Vesicles to Anemia Due to Inammatory Bowel
Disease in a Mouse Model
M Kohno
1
, H Horinouchi
1
, Y Izumi
1
, T Ikeda
1
, H Sakai
2
, E Tsuchida
2
, K
Kobayashi
1
1
Department of Surgery, Keio University School of Medicine, Tokyo Japan
2
Advanced Research Institute for Science and Engineering, Waseda University,
Tokyo, Japan; kohno@1993.jukuin.keio.ac.jp
Human inammatory bowel disease (IBD) is a chronic, relapsing and remitting
inammatory condition of unknown origin that aficts individuals of both sexes
throughout life. The disease is clinically characterized by two phenotypes
ulcerative colitis and Crohns disease. Anemia affects between 30%and 70%of
patients and has great impact on the quality of their lives. They currently receive
transfusion of red blood cells for severe anemia. The purpose of this study was
to assess the feasibility and safety of infusion of encapsulating concentrated
human hemoglobin (Hb vesicles) for treatment of anemia due to IBD. Infusion
of Hb vesicles did not exacerbate IBD as determined by a serum biomarker
and histologic evaluation compared with saline in the dextran sulfate sodium
mouse colitis model. Further, disease activity index for IBD and body weight
loss tended to be decreased by infusion of Hb vesicles.
SVI-4
Genetic Engineering of Heme Pocket in Human Serum Albumin: Control
of O
2
Binding of Iron Protoporphyrin Ix
T. Komatsu,
1,2
A. Nakagawa,
1
E. Tsuchida
1
1
Reserach Institute for Science & Engineering, Waseda University, Tokyo 169-
8555 Japan,
2
PRESTO, Japan Science and Technology Agency (JST), Saitama
332-0012 Japan; teruyuki@waseda.jp
Complexing an iron protoporphyrin IX into a genetically engineered heme
pocket of recombinant human serum albumin (rHSA) generates an articial
Oral Presentations 219
hemoprotein, which can bind O
2
in much the same way as hemoglobin (Hb).
1
We previously demonstrated a pair of mutations that are required to enable
the prosthetic heme group to bind O
2
reversibly: (i) Ile-142His, and (ii)
Tyr-161Phe or Leu [I142H/Y161F (HF) or I142H/Y161L (HL)].
2)
Here we
report additional new mutations designed to manipulate the architecture of the
heme pocket in rHSAheme complexes by specically altering distal amino
acids. We show that introduction of a third mutation on the distal side of the
heme can modulate the O
2
binding equilibrium.
3)
The HSA(HL/L185N)heme
showed very high O
2
binding afnity (P
O2
1/2
: 1 Torr, 22

C), which is 18-fold

greater than that of the original double mutant rHSA(HL)heme and very
close to the afnities exhibited by myoglobin and the high-afnity form of Hb.
Introduction of Asn at position 185 enhances O
2
binding primarily by reducing
the O
2
dissociation rate constant. Replacement of polar Arg-186 with Leu or
Phe increased the hydrophobicity of the distal environment, yielded a complex
with reduced O
2
binding afnity (P
O2
1/2
: 910 Torr, 22

C), which nevertheless

is almost the same as that of human red blood cells and therefore better tuned
to a role in O
2
transport.
SVII-3
Microcirculation Disorders Under Chronic Venous Insufciency: Diagnos-
tics and Correction with Taxifolin
VI Kozlov, VP Tihonov, LI Dergatchova, GAAzizov, OAGurova, VVBaranov
Department of anatomy and Department of laser medicine, Peoples Friendship
University of Russia; Company Diod; Company Centre for Analysis of
Substances, Moscow; Russia kozlov@med.rudn.ru
The purpose of the present research consisted to investigate the inuence
bioavonoid Taxipholin [Dihydroqercetin], possessing antioxydative action,
on a skin microcirculation at patients with chronic venous insufciency [CVI].
45 patients with CVI [CEAP stage 2 4] have been surveyed at treatment
of 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 3
weeks. Methods: The estimation of skin microcirculation in a leg and foot was
made by means of Laser Doppler owmetry [LDF] [device LAKK, Lazma-Co,
Rassia], and also TV-computer capillaroscopy of eponichii on 1 nger 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 are
revealed. Under biomicroscopy at them increases of permeability of their wall
and rheologic frustration were marked both structural changes of microvessels.
The degree of insufciency of microcirculation directly depend on weight of
current CVI. The leading pathogenetic mechanism in frustration of microcir-
culation are: progressive increase of the phenomena of venous stagnation on a
microvessel level, local blockade of a capillary blood ow and sharp damage
220 Oral Presentations
of barrier function of microvessel wall. The most vulnerable is a nutritive part
of microvascular network. Frustration of a blood ow at CVI lead to change
of myogenic activity of arterioles and precapillaries. At LDF it is expressed
in reduction of uxmotion amplitude, spectral narrowing of oscillations and
displacement of their dominant in high-frequency area that is a result of sup-
pression the vasomotion mechanism. Application of gel Taxipholin positively
affects dynamics of parameters of a local and systemic microcirculation at
patients CVI. After the lead treatment improvement of microcirculation and
diminish of stagnation tissue blood ow was observed: average linear speed of
erythrocytes 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 perivascular
zone [on 20 25%] decreased. Comparison of gel Taxipholin to gel Trox-
erutin [placebo] has shown, that at them comparable vasotropic effect. At the
same time it has noted been, that reactance of microvessels [on postural test]
at patients CVI it becomes authentic above [on 11%] after treatment by gel
Taxipholin. Conclusion: Taxipholin possesses vasotropic action at the level of
microvessels and owing to antioxydant action positively inuences on normal-
ization of microcirculation and decrease in permeability of capillary wall at
patients with CVI.
SII-2
Engineering Hemoglobin-Based Oxygen Carriers for Tissue
Engineering
Andre F Palmer
Department of Chemical and Biomolecular Engineering, The Ohio State Uni-
versity, Columbus, Ohio, USA; palmer.351@osu.edu
A priori knowledge of the dissolved oxygen (O
2
) concentration prole within
hepatic hollow ber (HF) bioreactors is important in designing and develop-
ing an effective bioarticial liver assist device (BLAD) for eventual clinical
use. O
2
transport is limiting within such HF bioreactors due to the poor sol-
ubility of O2 in aqueous culturing media, long tortuous O2 diffusion paths
within HF membranes, and high hepatocyte O2 consumption rates. We hy-
pothesize that supplementing a hepatic HF bioreactors circulating media
stream with a hemoglobin-based oxygen carrier (HBOC) will improve hepa-
tocyte oxygenation. Our results (experimental and theoretical) indicate HBOC
supplementation 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 results
thus support the use of HBOCs for improving oxygen delivery to hepato-
cytes maintained within HF bioreactors. In general, the results of this work
will be indispensable in improving oxygenation of bioreactors used for tissue
engineering.
Oral Presentations 221
SVI-2
Haemorheological Characterization of a Peruorocarbon-Based Oxygen
Carrier (Oxygent) in Microcirculatory Scale Flow
JP Peach
University of Vermont, Burlington, VT, USA; isbs2007spam@jppeach.com
Vascular (geometric and topographical) and intravascular (rheological) factors
inuence the resistance to blood ow. Addition of drugs, such as Oxygent, to
the blood may alter the haemorheology and therefore affect the cardiovascular
systems 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 of
this study were to characterize the rheological properties of Oxygent mixtures
and then evaluate its cardiac work load requirements in a physiologically rel-
evant numerical model. To do this, whole blood was diluted with Oxygent or
phosphate buffered saline 7.2 (PBS) to produce mixtures with 25, 30, 35 or
40% haematocrit and 60% plasma. A 100-m diameter glass-tube viscometer
was used to measure the pressure drop over a 32-mmlong test section at various
wall shear rates (100 1,800/sec). Viscosity was then computed and a regres-
sion analysis was used to develop equations which predict viscosity from the
shear rate for each haematocrit ratio group. To test the effects of temperature
on pure Oxygent, its viscosity was determined in the temperature range of 32.2
41.1

C with wall shear rates between 17.5 and 2,275/sec. Lower tempera-
tures led to signicantly (p < 0.0005) higher viscosity of Oxygent. In an effort
to interpret these results in a clinically relevant framework, cardiac workload
over a 50-segment vascular network was examined. The pressure drop over the
network was used as a measure of cardiac workload. The boundary conditions
(inlet pressure, owrate and haematocrit) were randomly selected for each inlet
segment. The pressure drop using an Oxygent-blood mixture was compared to
the pressure drop using a PBS-blood mixture having the same boundary condi-
tions. These results will provide a theoretical measure of the cardiac workload
required to pump an Oxygent-based blood mixture compared to a PBS- based
mixture.
SV-1
Hemoglobin-Vesicles as Articial Oxygen Carriers: Interactions with Lig-
and Molecules in the Production Process and in Blood Circulation
H Sakai
1
, A Sato
1
, K Sou
1
, H Horinouchi
2
, K Kobayashi
2
, E Tsuchida
1
;
1
Research Institute for Sci. & Eng., Waseda University, Tokyo, Japan;
2
Dept. of
Surgery, School of Medicine, Keio University, Tokyo, Japan; hiromi@waseda.jp
Hb-vesicles (HbV, 250 nm) are articial O
2
carriers, and the safety and efcacy
as a transfusion alternative have been claried in detail [17]. Hb binds not only
O
2
but also CO and NO very strongly. In some conditions HbCO dissociates
222 Oral Presentations
easily. The interactions of HbV with these gaseous ligands are important both
in the production process and in blood circulation. HbCO is resistant to heating
and it enables pasteurization at 60oC for 10 hrs to guarantee the utmost safety
from infection [8]. The puried HbCO is concentrated to ca. 40 g/dL and
encapsulated with a lipid bilayer membrane without protein denaturation. The
vesicular surface is decorated with PEG. HbCOcan be easily converted to HbO
2
by photoirradiation in an aerobic condition. Finally O
2
is completely removed
out and the resulting deoxy-state HbV can be stored at room temperature
for over 2 years [9]. HbV does not induce vasoconstriction in contrast to
molecular Hbs. This difference presumably relates to the reduced uptake of
endogenous NOand COowing to the large dimension and the cellular structure
of HbV [3,4]. Recently, we tested intravenous injection of exogenous CO-
bound HbV into rats, and found out that CO was released quite promptly in
3 hrs with a cytoprotective effect at reperfusion. This indicates a possibility
of a new clinical application of HbV in addition to its use as a transfusion
alternative.
PA-4
Accurate and Quantitative Determinatuion of the Structural Proles of
Hemoglobin Vesicle By Means of X-Ray and Light Scattering Techniques
T. Sato
1
, H. Sakai
1
, K. Sou
1
, O. Glatter
2
, E. Tsuchida
1
Research Institute for Science and Engineering, Waseda University, Tokyo,
Japan, and
2
Institute of Chemistry, University of Graz, Graz, Austria;
takaaki.sato@waseda.jp
The Waseda-type Hemoglobin vesicle (HbV), developed as a cellular-type ar-
ticial oxygen carrier encapsulating a solution of puried and concentrated
human hemoglobin (35 g/dL) with a surface-modied phospholipid bilayer
membrane 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 dispersion
evaluated with Optimized Regularization Technique have provided an accurate
quantitative estimation of the size and size distribution of HbV; we obtained
an 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 rst time made it possible to observe col-
lective diffusion process of a concentrated (10g/dL) HbV dispersion without
dilution, overcoming the interference from multiple scattering and absorption
caused by the hemoprotein. The result conrms an ergodic (uid-like) nature
and good stability of the HbV dispersion.
The forward SAXS intensity of HbV dispersion represents the feature of
a slightly polydisperse sphere having an averaged radius of 120 nm, whereas
the high q-part reects the internal structure of HbV, e.g., highly concen-
trated Hb (>35g/dL) and lipid bilayer. SAXS experiments have also revealed a
Oral Presentations 223
uni-lamellar structure, the thickness (5.8nm) and internal electron density pro-
le of the vesicle for Hb encapsulation.
SII-4
Hemotech, a Novel Free Hemoglobin-Based Red Cell Substitute with Phar-
macological Properties: the Concept and Current Status of Commercial
Development
J Simoni, M. Feola, G Simoni, JF Moeller, BT Mittemeyer, AP Bollon
HemoBioTech, Inc., Dallas, Texas, U.S.A. and Texas Tech University Health
Sciences Center, Lubbock, Texas, U.S.A; jan.simoni@ttuhsc.edu
The worldwide need for blood substitutes is evident; however, currently tested
free hemoglobin (Hb)-based oxygen carriers have toxicity and efcacy prob-
lems. These products were developed before the recognition of Hbs intrinsic
toxicity; therefore, it is not surprising that the commercial development of sev-
eral rst-generation products has been discontinued. The problems with these
blood substitutes revolve around blood vessel constriction and the pro-oxidant
and pro-inammatory properties of heme. To diminish intrinsic toxic effects of
Hb, Texas Tech University Health Sciences Center scientists have developed
and patented a novel concept of pharmacologic cross-linking and formulated
an 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 puried bovine Hb, cross-linked intramolecularly with
open ring adenosine 5

-triphosphate (o-ATP) and intermolecularly with open

ring 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-inammatory properties of Hb with the activation of adenosine
receptors, which would produce vasodilatation and reduce inammatory re-
actions. The concept of conjugation of Hb with GSH was to introduce more
electronegative charges onto the surface of Hb, which would block Hbs trans-
glomerular and transendothelial passage, and would make it less visible to
phagocytes. In addition, GSH shields heme from reactive oxygen species and
nitric oxide. The reaction with o-ATP stabilizes the Hb tetramer, but the re-
action with o-adenosine allows the formation of Hb low molecular weight
polymers with uniform electronegative charge. HemoTech was subjected to
preclinical testing and clinical proof of medical concept. The results of these
studies are favorable, indicating that HemoTech has vasodilatory activity and
can reduce vasoconstriction that follows hemorrhage, has erythropoietic ac-
tivity and produces no adverse nephrotoxic, neurotoxic, oxidative, inamma-
tory or apoptotic reactions. These ndings indicate that in order to design
a non-toxic and efcacious free Hb-based blood substitute product, pharma-
cologic cross-linking of the Hb molecule is necessary. Now, HemoTech has
entered the regulatory process for commercial development in the U.S. and
abroad.
224 Oral Presentations
PA-3
Effect of Hemospan
R
on Microvessel Diameter and Functional Capillary
Density in a Transgenic Mouse Model of Sickle Cell Anemia
AG Tsai
1,2
, P Cabrales
1
, MA Young
3
, RM Winslow
2,3
, M Intaglietta
2
1
La Jolla Bioengineering Institute, La Jolla;
2
Department of Bioengineering,
University of California, San Diego, and
3
Sangart Inc. San Diego, CA/USA;
agtsai@ucsd.edu
Targeting O
2
delivery to anoxic regions may reduce the incidence of com-
plete deoxygenation of the RBC and thus be an effective therapy in sickle cell
disease. Knockout transgenic mice (n = 6) were treated with an infusion of
Hemospan
R
, 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 of
microvessel diameter and functional capillary density (number of capillaries
perfused per area of tissue) were studied in vivo using the dorsal skin fold
window chamber model. Mean arterial pressure was higher after treatment
during normoxia and hypoxia challenge as compared to the control group. Ar-
terioles dilated and venules remained unchanged from baseline during hypoxia
in both study groups. FCD was statistically reduced from baseline during hy-
poxia; however Hemospan
R
infused animals maintained FCD at a level that
was statistically higher than observed with saline infusion; 60 15% and
27 24%, respectively. Thus Hemospan
R
treatment reduces the severity of
hypoxia-mediated decline in FCD. Mechanistically the higher FCD could be
due to the higher perfusion pressure in the absence of vasoconstriction. Re-
duced sickling and inammatory responses (i.e., lesser leukocyte-adhesion)
are likely concomitant mechanisms which support and enhance capillary
perfusion.
Supported by NIH BRP Grant HL064395.
SII-3
Design of A Platelet Substitute Utilising Host Fibrinogen to Enhance
Haemostatis
GF Walker
1
, J.A. Appleby
1
, SM Middleton
1
, AH Goodall
2
1
Haemostatix Ltd, BioCity, Nottingham, UK.
2
Cardiovascular Sciences, Uni-
versity of Leicester, UK; gregthekiwi@hotmail.com
A novel platelet substitute is being developed as a safe replacement for platelet
transfusion. The product is designed to bind host brinogen (Fgn) after injection
and interact preferentially with activated platelets at the site of a wound. The Fgn
binding peptide Gly-Pro-Arg-Pro (GPRP) was conjugated to human albumin
Oral Presentations 225
microparticles (MPs). FITC-labelled Fgn binding by the MPs was determined
by ow cytometry. To assess MP incorporation into a brin clot, GPRP-MPs
were added to platelet free plasma with thrombin (0.25 U/mL) and brin forma-
tion monitored in a Platelet Aggregation Proler. Impedance aggregometry was
used to assess MP interaction with unactivated and ADP-activated platelets in
whole 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-labelled
Fgn 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 into
the brin clot. Impedance aggregometry showed that GPRP-MPs in platelet-
depleted blood enhanced ADP (3 M)-induced aggregation compared with
control 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 control
MPs. (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 rabbits
receiving GPRP-MPs compared with control-MPs (9.3 5.5 vs 20.1 7.6
mL; p = 0.02). GPRP-MPs bind Fgn and exhibit haemostatic activity in vitro
and in vivo. Fibrinogen-free GPRP-MPs have potential as an effective platelet
substitute with signicant safety and production advantages.
SV-5
Blood Banking-Induced Impairment of Red Blood Cells Flow Properties
Saul Yedgar
1
, Alex Koshkaryev
1
, Hannah Relevy
2
, Nogah Manny
2
, Gregory
Barshtein
1
1
Department of Biochemistry, Hebrew University Medical School, and
2
Blood Bank, Hadassah University Hospital, Jerusalem, Israel; 91120.
yedgar@md.huji.ac.il
Background: Blood banking procedures are associated with damage to red
blood cells (RBC), which can impair their ow properties, namely their de-
formability, self-aggregability, and adherence to endothelial cells (EC) of blood
vessel walls. Transfusion of such RBC might thus introduce a circulatory risk
to recipients. The present study was undertaken to comprehensively explore
the effect of cold storage and -irradiation on RBC ow properties. Methods:
RBC ow properties were monitored prior to and during storage, as well as
before and after -irradiation, as a function of shear stress, using a comput-
erized cell owproperties analyzer. Results: Routine cold-storage markedly
decreased RBC deformability, and strongly elevated their self-aggregability
and their adherence to EC, as expressed by the amount of altered RBC and
the strength of intercellular interactions. These changes were observed already
at the second week of the storage period. The elevation of RBC/EC interac-
tion was well correlated with translocation of phosphatidylserine to the RBC
surface. -Irradiation induced an immediate, sharp increase in the number of
226 Oral Presentations
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 ow properties facilitate circulatory disor-
ders, the potential circulatory risk of the practiced blood banking procedure
should 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-1
Peg-Conjugated Hemoglobin (Hemospan
R
) Liganded with Carbon
Monoxide Reduces Myocardial Infarct Size Following Ischemia /Reper-
fusion in Rats
Mark Young, Jeff Lohman, Ashok Malavalli, Kim Vandegriff, Robert Winslow
Sangart, Inc. San Diego, CA, USA; lbarkley@sangart.com
Carbon monoxide (CO) is reported to confer cytoprotective effects in models of
hypoxia, and ischemia. However therapeutic strategies are complicated by the
lack 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 with
CO (CO-MP4) in a rat model of myocardial ischemia and reperfusion, and
compared the effects with oxy-MP4 or -Hb. Lactated Ringers (LR) and
preconditioning (PC) were employed as negative and positive controls, respec-
tively. Pentobarbital-anesthetized rats were mechanically ventilated (FiO
2
=
0.3) and subjected to 30 min of coronary ligation followed by recovery and
24 hr reperfusion. Treatment (30% blood volume topload) was begun prior to
ligation and continued for 24 hr. Non-ischemic area (area at risk, AAR) was
distinguished by Evans blue and infarct size (IS) was delineated with triph-
enyl tetrazolium chloride. Ischemic area was uniform in all animals as AAR
did not differ between groups. Infarct size (IS/AAR) was reduced (P <.05) in
CO-MP4 animals (37 6%) compared with oxy-MP4 (61 7%), -Hb (63
5%), or LR (53 3%). PC elicited the greatest protection (IS/AAR = 13
6%). CO-Hb at the end of ischemia was 5.20.2% of total Hb in CO-MP4
treated rats. Arterial pressure and heart rate did not differ between groups of
rats. In separate studies with rats, CO was found to distribute equally between
MP4 and RBC-Hb within 20 min, and disappear from the blood within 30 min
following cessation of infusion. These data demonstrate that CO is cardiopro-
tective in this model, and that MP4 may be a safe and controlled method of
delivery.
SVII-4
Effect and Mechanism of PEG-conjugated Hemoglobin on Hypoxia Mi-
croenvironment in HeLa Tumor Model
Oral Presentations 227
Min Dai
1
, Minghua Yu
1
, Jianqun Han
1
, Hongwei Li
1
, Jian Zhang
1
, Qian Liu
2
,
Ruijuan Xiu
1
1Institute of Microcirculation, Chinese Academy of Medical Sciences (CAMS)
& Peking Union medical college (PUMC),
2
Peking Union Medical College
Hospital (PUMCH), Beijing, China; xiurj@yahoo.com.cn
Tumor hypoxia microenvironment is strongly associated with a diminished
therapeutic response and malignant progression. So it is then not surprising
that hypoxia has been considered an attractive target for the development of
novel anti-cancer therapies. A number of articial oxygen carriers are cur-
rently in advanced clinical trials for their ability to replace red blood cells and
to ensure adequate tissue oxygenation. However, little has been done to inves-
tigate that if intravenous administration of blood substitutes were effective in
increasing the oxygenation throughout experimental tumors. To investigate the
effect 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 a
week, and then the animals were sacriced after a month. Hypoxyprobe
TM
-1
(pimonidazole hydrochloride) was used the detected the Oxygen gradients in
tumor tissue. ELISA and immunocytochemistry methods used for the detec-
tion of HIF and VEGF protein in blood and tumor tissue separately have been
determined. The results showed that hypoxia in tumor tissue were attenuated
by PEG-conjugated hemoglobin solution. HIF and VEGF expression were de-
cline compared with the control group. Taken together, our data show that
PEG-conjugated hemoglobin suppresses the HIF and VEGF expression due to
the increase of tumor tissue oxygenation.
SIV-1
The Study on a Rat Exchange Transfusion Model By Articial Red Blood
Cells (Hemoglobin Vesicle)
Jing Fan, Xueqiao Wang
Tianjin Blood Center Tianjin, P.R. China; fanjing668@eyou.com
Objective: In a rat exchange transfusion model,as an articial oxygen carrier,
the security and validity with the articial red blood cells (hemoglobin vesicle,
HbV) has been veried. Methods: The rst, to construct a best rat exchange
transfusion model. The second, using the model to transfuse blood with the
articial red blood cells. The nally,to evaluate the capabilities of the articial
red blood cells by some methods (such as: Blood routine test, Blood gas
test, Flow cytometry and Pathology), and to compare the therapeutic effect
of the different product in the rat acute hemorrhagic shock and resuscitation.
Results: The articial red blood cells (HbV) has been developed to provide the
oxygen-carrying ability. The Studies have shown that after approximately 70%
of hemoglobin is lost from circulation, then supply the articial red blood cells
228 Oral Presentations
suspension,the rats survive rate is 100%. The Pathology results indicate that no
signicant change in the viscera(heart, liver, kidney, lung). Conclusion: The
articial red blood cells (HbV) are the articial oxygen carrier with bionics
meaning. It can be helpful the acute hemorrhagic shock rats pass the rst hour
after the severe trauma, that is golden hour.
Key words: articial red blood cells; hemoglobin vesicle; a rat exchange
transfusion model
SVII-1
Effect of PEG-Conjugated Hemoglobin Solution with Chemotherapy on
characteristics of tumor neovascularization
Jianqun Han
1
, Min Dai
1
, Minghua Yu
1
, Hongwei Li
1
, Jian Zhang
1
, Qian Liu
2
Ruijuan Xiu
1
1
Institute of Microcirculation, Chinese Academy of Medical Sciences (CAMS)
& Beijing, China Peking Union Medical College (PUMC),
2
Peking Union
Medical College Hospital (PUMCH) Beijing, China; xiurj@yahoo.com.cn
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 notable
characteristics of newly formed tumor vessels which conversely worsen tumor
tissue oxygen provision. The aim of this study was focused on the tumor mi-
crovessels morphology changes when PEG-conjugated hemoglobin combined
with cislplatin treatment. Inoculated tumor cells to the submucosa of golden
hamsters cheek pouch to establish tumor model. Computer assisted intravital
microscopy was used to observe and measure microvessels tortuosity and func-
tional capillary density. At 5 days post-innoculation microvessel tortuosity close
to tumor mass was signicantly decreased in animals received both cisplatin
and PEG-Hb (0.6 g/kg) compared to animals received single agent cisplatin.
However, no signicant changes was seen in animals received both cisplatin
and PEG-Hb (0.3 g/kg) compared to control. Similar tendency was presented in
tumor functional capillary density between the two groups. The improvement
of newly formed vessel structure was positive corelation with tumor tissue
oxygenation. Conclusion: Higher dose of PEG-conjugated hemoglobin solu-
tion with chemotherapy treatment is benet to tumor tissue oxygenation and
microvessel normalization. Improved tumor vasculature facilitate oxygen and
chemotherapic agents transport to tumor tissue. It is indicated that the evaluated
PEG-conjugated hemoglobin may be a potencial adjuvant to chemotherapy in
cancer.
SVI-3
Polyoxyethylene-Modied Albumin-Heme Hybrid Synthesis, Property
and Oxygen-Binding Ability
YB Huang
1
, T Komatsu
2
, E Tsuchida
2
Oral Presentations 229
1
State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute
of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, Jilin
Province, P.R.China.
2
Advanced Research Institute for Sciences and Technolo-
gies, Waseda University, Tokyo, Japan; ybhuang@ciac.jl.cn
Recombinant human serum albumin (rHSA) incorporates a synthetic heme,
providing an articial hemoprotein [albumin-heme (rHSA-heme)] which has
the potential to bind and release f under physiological conditions (pH7.3, 37

C)
in the same manner as Hb.
In order to improve the circulation persistence in blood stream, we have
recently prepared a new albumin-heme hybrid modied by polyoxyethylene
(PEG) derivatives, and its structure, property and oxygen-binding ability were
carefully investigated. The maleimido-activated or succinimide-activated PEG
(Mw. 2 kDa and 5 kDa; M2000, M5000, S2000 and S5000) were used for
covering the surface of rHSA-heme by covalently bond.
By controlling the reaction conditions, 4 types of PEG -modied rHSA-
heme ([rHSA] 5 wt%) with 6 PEGchains have been synthesized; numbers of the
chains were determined by MALDI TOFMS analyses. The PEG(rHSA-heme)s
also bind O
2
reversibly dependent of the O
2
partial pressure. The O2-binding
afnities (p1/2) were 3136 Torr and the half times of the oxy-form were
around 12 hr (37

C). The viscosity of the S5000 and M5000 modied PEG

(rHSA-heme) solutions showed relatively high values, while the viscosity of
S2000 and M2000 modied PEG (rHSA-heme) were nearly the same as that
of the rHSA-heme solution. The circulation persistence of the heme was also
investigated by 20% top-load in rats, and the half lifetime was signicantly
extended 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-transfusion
project.
SIII-2
Modication of Hemoglobin by Crosslinking with a Band 3 Terminus
Derived Peptide as a Blood Substitute
YL Lin, KT Huang
Department of Chemical Engineering, National Chung Cheng University, Chia-
Yi, Taiwan, China; d92425001@ccu.edu.tw
Hemoglobin solution lacking 2,3-diphosphoglycerate (2,3-DPG) is not able
release sufcient oxygen in the circulation of human body. In this study,
therefore, a peptide composed of 9 amino acids, 7 from N terminus of
human erythrocytic Band 3 protein (AcMEELQDD) and cysteine and glu-
tamic acid added sequentially in C terminus is crosslinked to hemoglobin
230 Oral Presentations
surface serving as a dynamic allosteric effector of hemoglobin for oxygen
release. The derivative of polyethylene glycol (PEG), Maleimide-PEG-N-
hydroxysuccinimidyl (MAL-PEG-NHS) was used to crosslink hemoglobin
with peptide. The crosslinked peptide on hemoglobin is able to modulate the
oxygen afnity of hemoglobin, resulting in a higher P
50
value of this peptide-
modied hemoglobin (peptide-PEG-Hb) in the oxygen dissociation curve as
compared to unmodied hemoglobin. Furthermore, succinimidyl propionate-
PEG-succinimidyl propionate (SPA-PEG-SPA) was used to crosslink intra-
and inter-hemoglobin molecules to prevent the dissociation of hemoglobin
tetramers. This resulting hemoglobin polymer exhibited a similar oxygen dis-
sociation behavior to peptide-PEG-Hb. Taken together, we have successfully
modied hemoglobin with a conjugated oxygen afnity modulator providing a
new aspect of hemoglobin-based blood substitute.
SVII-2
Effects of Different Molecular Weight of Hydroxyethyl Starch on Hemor-
rhagic Shock in Rats
1,2
Li Tao,
2
Liu Liang-ming,
1
Yang Chengmin
1
Tianjin Concord Biotech De-
velopment Research Institute
2
State Key Laboratory of Trauma, Burns and
Combined Injury, Department 2
Research Institute of Surgery, Daping Hospital, The Third Military Me-
dial University, Chongqing 400042, China smalldiablo@gmail.com, cheng-
minyang2602@163.com
Objective: To compare the resuscitation effect of 6% different molecular
weight 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 mean
arterial blood pressure(MAP), left intraventricular systolic pressure(LVSP), the
maximal change rate of left intraventricular pressuredp/dt
max
) of hemorrhagic
shock rat were observed. Meanwhile, the artery blood gas, the survival time
and 24 hour survival rate were also observed in the present study. Results:
Improving hemodynamic parameter effects of our HES on hemorrhagic shock
rats were similar to Voluven and Haes. HES130 and HES200 prolonging the
survival time of shocked animals was pretty better than HES40, among the
three types of molecular weight HES, HES200 had the best effect and equive-
lant to Voluven and Haes. Conclusion: Three types of molecular weight HES
produced by our company have good benecial effect on hemorrhagic shock.
HES200 has a better effect.
Key Words: Hemorrhagic shock; HES; Voluven; Haes
SIII-5
Porphyrin-Histidine System Recognition of CO2 and Other Anions:
mimetic of hemoglobin action in physiological conditions
Oral Presentations 231
Di Li
1
, Wenqian Dong
1
, Tianjun Liu
2
Chinese Academy of Medical Sciences & Institute of Biomedical Engineering,
Peking Union Medical College, Tianjin 300192, China; merthonl@hotmail.
com
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 moves
the life cycle in animals and human beings. As an important research topic
in biomaterials, with the aim to mimetic and substitute natural system, there
are a lot of research papers focused on the oxygen carrier system all over the
world, such as human and animal hemoglobin modication, natural or syn-
thetic heme-based oxygen carriers, recombinant and transgenic hemoglobin as
well as peruorocarbon based oxygen carriers, etc. All of these reveal that oxy-
gen carrier is very important. However, the action of carbon dioxide has been
ignored, and less reports pay attention to the carbon dioxide, which plays an
almost 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 lethal
for the patient suffering of lung illness. Here we design and synthesize some
model compounds with porphyrin as platform with histidine as binding point,
and try to mimetic the hemoglobin acting on carbon dioxide in physiological
conditions and other systems.
In porphyrin chemistry,molecular recognition has been developed rapidly
and turned out to be a front topic in recent years. As a favorable host, por-
phyrin can recognize multifarious ion and bioactive molecule, which could
be used in the eld of enzyme analogue, phototherapy, molecular probe and
so on. Here in this paper the following content was reported : 1. Synthesis
of porphyrin-histidine system: a series of porphyrin-histidine compounds
were synthesized with either free amino groups or free imidazole groups or both
as binding points. 2. Evaluation of molecular recognition ability for different
compounds: The effect of functional groups such as amino or imidazole group
as well as ambient environmental effect on molecular recognition was studied.
Results showed that molecular recognition ability correlated tightly with the
compound structure of host and guest. For the same host, different binding
ability to HCO3-, CO32-, H2PO4-, HPO42-, SO42-, SO32- were detected. 3.
Comparing the absorbing CO2 ability for different compounds: On the basis
of pressure balance, a simple equipment was designed to detect absorption of
carbon dioxide. Results showed that free amino group plays a predominant
role in carbon dioxide absorption, while imidazole moiety acts less in our
system.
Summary: the synthetic porphyrin-histidine show the selective recogni-
tion ability for different anions, and its binding ability depends on both stere-
ospecic blockade and chemical structure. The absorption of carbon dioxide
of these system benets from the free amino group in the porphyrin-histidine
system.
232 Oral Presentations
SIII-1
Protection of Isolated Eat Heart by Polyethylene Glycol-Bovine
Hemoglobin Solution
X Chang, C Long
Fuwai Hospitai, Beijing, P.R. China; changxin718@yahoo.com.cn
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, on
the protection of isolated rat hearts. Rat hearts were harvested to transferred
to 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 results
showed that after reperfusion, cardiac function and histological change were
better in PEG-bHb groups, myocardial ATP contant was higher and cardiac
troponin I in coronary ow was lower in in PEG-bHb groups. This study
demonstrated that PEG-bHb in cardioplegic solutions can provide better my-
ocardial protection during ischemia.
SIV-2
Diffusion Behavior of Hemoglobin-Loaded Nanoparticles with Porous
Structure as Oxygen Carriers
Y Sheng, Y Yuan, CS Liu
Engineering Research Center of Biomedical Materials Under Ministry of Edu-
cation, East China University of Science and Technology, Shanghai, P.R. China;
sh-crystal@163.com
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 behaviors
of small molecules (such as glucose, ascorbic acid and glutathione) through
HbP were investigated. The results show that HbP with higher encapsulation
efciency (80%) and desirable diameter (70-200nm) were produced through
an improved double emulsion method; different molecular weight of PEGs,
coencapsulated in the internal phase with Hb, successfully detected the porous
structure of HbP, especially the pore size which it is difcult to nd a popular
apparatus to observe directly; meanwhile, distinct diffusion behaviors of small
molecules, which was dominated by the pore parameter of HbP, were obtained
based on various preparation conditions. It is signicant to understand the rela-
tionship between porous structure and diffusion behavior so that the preparation
of hemoglobin-loaded porous nanoparticles can be controlled, monitored and
reproduced with a desired diffusion prole of small molecules.
Many diseases are caused by tissue hypoxia that due to blood circulation
obstacles, which is the most serious problem in clinics. Prof. Yang studied
many drugs, such as oxygen carrying anti-shock drugs, the treatments drugs of
Oral Presentations 233
ischemic cardiocerebrovascular diseases and so on in recent years, the reports
of preliminary studies on the pharmacodynamics is below: Our preliminary
results showed that poly-human hemoglobin solution has capacities of oxygen
delivery together with remaining red blood cells, indicating potential clinical
applications in improving or curing microcirculation disorder. Experiments
of perfusion and preservation of isolated organs demonstrated that our prod-
ucts could signicantly protect hearts from ischemia/reperfusion injury and
appear improving the function of rat hearts after prolonged storage. We also
study blood ow in Golden Hamsters microcirculation. 13 male hamsters
were randomly divided into two groups. Control group include 5 hamsters with
ACD anticoagulant fresh blood of health people and Experimental group in-
clude 7 hamsters with human polymerized hemoglobin The whole exchange
transfusion volume was up to 40% and each mouse was completed within
15 minutes. Slow blood ow, stagnation, dyspnea, systemic convulsion and
nally asystole appeared immediately in ve mice of control group during
the period of exchange transfusion. Five mice were all dead within twenty
minutes. But only one mouse of the experimental group showed abnormal
reactions and was dead after 30 minutes. The remaining six mices microcircu-
lation kept well and all survived. In short, preliminary study demonstrates that
poly-hemoglobin solution has potential clinical application in improving or
curing disease of microcirculation disorder and perfusion and preservation of
isolated organ.
SIV-5
Reconstruction of Erythrocyte and Its Cellular Mechanical Properties
Xiang Wang, Wei Gao, Li Yang, Wei-yan Peng, Jia-xin Xie
College of Bioengineering, Chongqing University, Chongqing P.R.China;
xwangchn@vip.sina.com
Erythrocyte shape and its biomechanical properties have close relation to its
function. 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. The
experimental results indicated that the reconstructed erythrocytes were similar
to the natural erythrocyte: having biconcave disc shape, good deformability
and carrying-releasing oxygen function. The study implied the physiological
function of reconstructed erythrocyte were similar to that of natural erythrocyte.
SIII-6
Effect of Polymerization Reaction on Cation Exchange Chromatography
onMolecular Weight Distributionof PolymerizedHemoglobin-BasedOxy-
gen Carriers
X Wang, L Huang, CM Yang, JF Wang
234 Oral Presentations
Tianjin University; Tianjin, P.R. China, Institute of Tianjin Uion biotechnology
development company, Tianjin, P.R. China, Tianjin Polytechnology University,
Tianjin, P.R. China; tpuwx@sohu.com, chengminyang2602@163.com
Blood 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 that
the 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 specicity, leading
to uncontrolled polymerization. For these reasons, a new method, polymeriza-
tion reaction on cation exchange chromatography (PRCEC), is established to
provide control of cross-linking (Mw). When Hemoglobin is modied or poly-
merized by glutaraldehyde, the magnitude of positive charge on the molecule
reduces. Because the strength of binding depends on the size of charge car-
ried on molecules in cation exchange chromatography (CEC), hemoglobin or
polyHb with low modication or polymerization is enriched. If the polymer-
ization takes place in this asymmetrical system, they have the more reaction
odd than hemoglobin (polyHb) with high modication (polymerization). So,
polyHb with narrow molecular weight distribution and low average molecular
weight 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 two
tetramers (128kD) to four tetramers (256kD) with the yield of about 53%.
S1-4
Dextran-Hemoglobin
J. Tze-Fei Wong
Department of Biochemistry and Applied Genomics Center, Hong Kong
University of Science & Technology, Clear Water Bay, Hong Kong, China
bcjtw@ust.hk
Dextran-hemoglobin (DxHb) consists of a covalent conjugate between human
hemoglobin and dextran MW20,000. It was the rst blood substitute to allow a
complete red blood cell replacement in an exchange transfusion model followed
by spontaneous recovery under room air (1). It is equally effective in the
hemorrhagic shock model (2). Its function as an HBOC is conferred with the
following 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
efciency of cancer radiation therapy (4).
Oral Presentations 235
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-1
Inhaled Nitric Oxide Prevents Vasoconstriction after Tetrameric
Hemoglobin Infusion
Binglan Yu
1
, Michael J. Raher
1
, Rong Liu
1
, Kenneth D. Bloch
1,2
, Fumito
Ichinose
1,2
, Warren M. Zapol
1
1
Department of Anesthesia and Critical Care,
2
Cardiovascular Research Cen-
ter, Massachusetts General Hospital, Harvard Medical School, Boston, MA,
U.S.; 02114 byu1@partners.org
Background: Hemoglobin (Hb)-based oxygen carriers (HBOCs) have long
been 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). The
objective of our study was to investigate whether pretreatment with inhaled
NO could prevent the systemic hypertension caused by infusion of autologous
tetrameric Hb solution in mice. Methods: Tetrameric Hb solution (4 g/dl) was
prepared fromwhole blood of C57BL/6 mice, and administered IV (0.012 ml/g
BW) following a period of breathing either air or NO gas (80 ppm). Systolic
blood pressure (SBP) was measured non-invasively in awake mice by tail-cuff
method, and in anesthetized mice by invasive hemodynamics. Results: After
administration of tetrameric Hb, SBP increased from118 3 to 143 7 mmHg
(p < 0.05) in mice breathing air alone. In contrast, when mice were pretreated
with inhaled NO (80 ppm, 15 min), subsequent administration of tetrameric
Hb while breathing air did not alter SBP (118 3 vs. 120 2 mmHg, p =
NS). These ndings were conrmed using invasive hemodynamic methods.
Breathing NO prior to tetrameric Hb administration did not increase the metHb
level in plasma (4 1% at 10 min after administration). However, 80 ppm NO
breathing continued for 10 min after tetrameric Hb administration, markedly
increased plasma metHb levels (74 10%). Conclusions: Pretreatment of
mice with 80 ppm iNO for 15 min prevents the systemic vasoconstrictor effects
of subsequent IV tetrameric Hb infusion without causing methemoglobinemia.
SIV-4
Inuence of PEG-conjugated Hemoglobin on Tumor Oxygenation and
Response to Chemotherapy
236 Oral Presentations
Minghua Yu
1
, Jianqun Han
1
, Min Dai
1
, Hongwei Li
1
, Jian Zhang
1
, Qian Liu
2
,
Ruijuan Xiu
1
1
Institute of Microcirculation, Peking Union Medical College (PUMC) & Chi-
nese Academy of Medical Sciences (CAMS), Beijing, China;
2
Peking Union
Medical College Hospital (PUMCH), Beijing, China xiurj@yahoo.com.cn
The presence of hypoxia in solid tumors has been recognized for more than
50 years. Hypoxic tumors are signicantly 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 improving
oxygen supply either by oxygen or carbogen breathing or by increasing the
hemoglobin level and oxygen delivery. The use of articial oxygen carriers
represents 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.) could
improve the tumor oxygenation and to determine whether preadministration of
PEG-conjugated hemoglobin could enhance the anti-tumor efcacy of cisplatin.
Hypoxia marker pimonidazole staining was employed to detect tumor tissue
oxygenation status and treatment efcacy was determined by measurements of
tumor volume, tumor growth delay and the extent of tumor apoptosis. We found
that The application of higher dose (0.6 g/kg) PEG-hemoglobin solution could
signicantly ameliorate the hypoxic condition in cervical carcinoma xenograft
models. Co-administration of PEG-conjugated hemoglobin (0.6 g/kg) with cis-
platin produced signifcant tumor growth inhibition and a reduction in tumor
vasculature 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 still
needs to be carried out to minimize its potential toxicities.
SIV-3
Reduction and Suppression of Methemoglobin Loaded In the Polymeric
Nanoparticles Used As Blood
Substitutes
XL Zhang, YY, CS Liu
Engineering Research Center for Biomedical Materials of Ministry of
Education, East China University of Science and Technology, Shanghai;
zxlpsh@126.com
A novel nonenzymatic reduction and suppression route, combination a fast
pre-reduction by sodium dithionite (SD) and a sustaining post-reduction by
reducing 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 inuences of the preparation factors were also
optimized. In the pre-reduction, the metHb in the rawHb could be reduced from
over 90% to 1.2% using SD combination with gel ltration. In double emul-
sion preparation, lower emulsion strength, PEGylated polymer and addition
of miscible solvent, such as acetonitrile could pronouncedly suppress metHb
formation. The resultant metHb level in HbP under the optimal conditions was
about 5.6%, which can be further reduced to 1.4% by the reducing agents in
plasma with the help of superoxide dismutatse and catalase, named as the sus-
taining post-reduction. The results achieved are promising for the fabrication
of blood substitutes with controlled metHb level, which can fulll the function
of binding/delivering oxygen to tissues in vivo for future trials.