ARTICLE
Conjugation of Methoxypolyethylene Glycol
to the Surface of Bovine Red Blood Cells
Sharon I. Gundersen,y Andre F. Palmerz
Department of Biomolecular and Chemical Engineering, University of Notre Dame,
182 Fitzpatrick Hall, Notre Dame, Indiana 46656; telephone: 574-631-4776;
fax: 574-631-8366; e-mail: [email protected]
Received 5 June 2006; accepted 30 August 2006
Published online 28 September 2006 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.21204
ABSTRACT: Methoxypolyethylene glycol (mPEG) covalently bound to the surface of human red blood cells
(hRBCs) has been shown to decrease immunological recognition of hRBC surface antigens (Bradley et al., 2002).
However, there is an increasing shortage of hRBC donations,
thus making hRBCs scarce and expensive (Davey, 2004;
Riess, 2001). The goal of this study is to similarly PEGylate
the surface of bovine RBCs (bRBCs) with the aim of reducing the demand on human blood donations needed for
blood transfusions. This study investigates the feasibility of
modifying the surface of bRBCs with the succinimidyl ester
of methoxypolyethylene glycol propionic acid (SPA-mPEG)
for use as a potential blood substitute. The oxygen binding
affinity of PEGylated bRBCs was moderately increased with
increasing initial SPA-mPEG concentrations up to 4 mM
when reacted with bRBCs at a hematocrit of 12%. Oxygen
transport simulations verified that SPA-mPEG conjugated
bRBCs could still transport oxygen to pancreatic islet tissues
even under extreme conditions. PEGylated bRBCs reconstituted to a hematocrit of 40% exhibited viscosities on the
order of !3 cp, similar to hRBCs at the same hematocrit.
Taken together, the results of this study demonstrate the
success of PEGylating bRBCs to yield modified cells with
oxygen binding, transport and flow properties similar to that
of hRBCs.
Biotechnol. Bioeng. 2007;96: 1199–1210.
! 2006 Wiley Periodicals, Inc.
KEYWORDS: methoxypoly(ethylene glycol); stealth erythrocytes; bovine red blood cells; transfusion
Introduction
Due to the large variety of blood transfusion needs, blood is
in very high demand with an average of 38,000 units (1 unit
!450 mL) of human red blood cells (hRBCs) being used
every day in the US, supplied solely by volunteer blood
donations (Riess, 2001; Scott et al., 1997b). Threat of
disease transmission, complex donor screening process, and
costs of storage and handling of blood has resulted in a
decreasing US blood supply with a predicted shortage of
approximately 4 million units by 2030 (Riess, 2001). Thus,
there is a very high demand for a sterile, cost-effective, and
universally acceptable alternative to current allogeneic
hRBC transfusions.
Xenogenic blood from cows could provide a stable blood
supply from a controlled and monitored source. However,
the major restriction to xenotransfusion would be possible
immune recognition of surface antigens on the surface of
bovine RBCs (bRBCs). Hence, the surface of bRBCs would
have to be modified to overcome this immunological
barrier.
Research has been ongoing to test the feasibility of
modifying the entire surface of hRBCs to produce an
immunologically silent RBC by either removing surface
antigens or hiding them to prevent antibody binding
(Blackall et al., 2001; Scott, 1997a). Unfortunately,
removing surface antigens by enzymatic cleavage has been
shown to compromise the RBC’s structural and functional
integrity (Blackall et al., 2001). Coating the RBC membrane
to hide surface antigens is one potential strategy to preserve
the structural integrity of the RBC. Poly(ethylene glycol)
(PEG) has the potential to mask RBC antigens due to its
non-toxic and non-immunogenic characteristics (Jo and
Park, 2000; Scott et al., 2000). PEG conjugation neither
denatures RBC surface proteins nor influences diffusion and
mass transfer of small molecules such as oxygen (Bradley
et al., 2002; Scott et al., 2000). PEG has been used extensively
to modify surfaces, and it has many unique properties that
prevent protein and bacteria adsorption on surfaces such as
its hydrophilicity, flexibility, large exclusion volume in
water, and is sterically bulky providing a polymer cloud
around modified molecules or cells (Holmberg et al., 1993;
y
Graduate Student.
Assistant Professor.
Correspondence to: A.F. Palmer
z
! 2006 Wiley Periodicals, Inc.
Biotechnology and Bioengineering, Vol. 96, No. 6, April 15, 2007
1199
Jo and Park, 2000). PEG has also been shown to prevent
adsorption of IgG (a protein involved in activating the
complement system), fibrinogen (common plasma protein),
and the Streptococcus bacterium onto surfaces (Holmberg
et al., 1993).
The unique physical properties of PEG have inspired
investigations on the effects of covalently binding PEG to the
RBC membrane. Numerous studies have conjugated
methoxypolyethylene glycol (mPEG) to the hRBC membrane (mPEG-hRBCs), in order to camouflage hRBC
surface antigens (Bradley et al., 2002; Murad et al., 1999;
Scott et al., 2000). Previous studies demonstrated that
conjugating mPEG to the hRBC membrane (PEGylation)
had almost no effect on the hRBC’s structure and function.
These mPEG-hRBCs were reported to have the same oxygen
binding properties as unmodified hRBCs and their
deformability was unaltered. Although the chemical
cross-linker on the mPEG molecule usually binds to the
surface protein involved in anion transport, mPEG
modification did not affect cellular ion concentrations
(Bradley et al., 2002).
Several studies have shown that PEGylation of hRBCs
alleviates potential immune responses by preventing cell–
cell interactions required for RBC aggregate formation and
inhibiting antibody binding (Armstrong et al., 1997; Bradley
et al., 2002). Modification of hRBCs with 5 kDa mPEG
exhibited a decreased response by anti-sera to the A antigen
compared to unmodified hRBCs (Scott, 1997). However,
further studies by Bradley et al. (2002) showed that larger
mPEG molecules provided longer in vivo RBC survival.
Bradley attributed this behavior to the larger mPEG
molecule’s ability to immunocamouflage larger surface
proteins such as the Kidd protein, involved in urea transport
(Bradley et al., 2002; Garratty et al., 2002). Bradley et al.
(2002) also found that 20 kDa mPEG was sufficiently large
enough to camouflage Kidd blood group antigens.
However, conjugating the surface of hRBCs with mPEG is
still dependent on the collection of blood from human
donors. One possibility of creating a RBC substitute for use
in humans would involve conjugating the surface of bRBCs
with mPEG (mPEG-bRBCs). Though pigs are considered
the best possible source for organ and tissue transplantation,
cows provide a more suitable source for blood donations,
due to their larger blood volume and easier vein access
(Johnstone et al., 2004; Otchet, 2001; Williams, 1996). In
addition, bRBCs are more mechanically robust, and have
better osmotic stability compared to porcine RBCs.
In this study, mPEG was conjugated to the surface of
bRBCs, similar to previous conjugations to hRBCs, in an
attempt to create a potential non-immunogenic RBC. Thus
far we have examined the physical effects of modifying
bRBCs with varying concentrations of 20 kDa mPEG to
identify optimum reaction conditions that yield a functional
RBC. The physical properties of mPEG-bRBCs should be
similar to hRBCs, in order to produce effective hRBC
substitute with substantial in vivo half-life. Since the bRBC is
smaller in size compared to a hRBC (diameter !5.8 mm)
1200
and considered a foreign object, it is not expected to last past
the 60–90 day half-life of transfused hRBCs (Biopure, 2004).
We predict that the circulation half-life of mPEG-bRBCs
will be greater than current LEHb based blood substitutes
(>60 h) (Biopure, 2004; Phillips et al., 1999) with clearance
mediated through the reticuloendothelial system.
Materials and Methods
Bovine Red Blood Cells
This study used bRBCs purchased from Quad Five (Ryegate,
MT). After purchase all bRBC samples were washed three
times in PBS (pH ¼ 7.2) by centrifuging at 950 rpm for
!30 min, discarding supernatant, and resuspension in PBS
three times. Once washed bRBCs were suspended to 40%
hematocrit. Quad Five carefully selects and quarantines their
donor animals. All animals are maintained under veterinary
care and diagnostic protocols, and bled in separate facilities
to maintain quarantine conditions. The cattle are routinely
screened for tuberculosis, brucellosis, blue-tongue virus,
anaplasmosis, leptospirosis, IBR, P13, BVD, and bovine
leukosis virus (Micks, 2005).
mPEG Derivative
The 20-kDa succinimidyl ester of methoxypolyethylene
glycol propionic acid (SPA-mPEG) was purchased from
Nektar Therapeutics (Huntsville, AL). The succinimidyl
ester moiety mPEG was designed for protein surface
modification and was recommended by Nektar for this
purpose (Harris and Kozlowski, 1995; Nektar Therapeutics,
2006). These linear monofunctional polymers are capped on
one end by a methoxy group to produce products free of
cross-linking (Nektar, 2006). The N-hydroxysuccinimide
active ester on SPA-mPEG binds to amino acid residues with
amines (such as lysines) producing a stable amide link. Since
mPEG is covalently linked to surface proteins of bRBCs, and
is free of hydrolysis-prone ester linkages, it is unlikely that
mPEG will elute or fragment in vivo.
mPEG Derivatization
bRBCs were diluted to 12% hematocrit in PBS (pH ¼ 7.2)
prior to reaction. Solutions of mPEG were prepared at
varying concentrations, 0 mM (buffer control) to 4 mM in 1
mM increments in 50 mM K2HPO4 and 105 mM NaCl
(mPEG buffer). An additional unreacted control of bRBCs
mixed with PBS was also evaluated to determine if the
reaction procedures had any effect on the bRBCs. Bovine
erythrocytes and mPEG solutions were mixed in equal
volumes, and allowed to react for 1 h at room temperature
and pressure on a 3-D rotator as described previously
(Bradley et al., 2001, 2002). At the end of the reaction,
modified bRBCs were washed three times in PBS and
Biotechnology and Bioengineering, Vol. 96, No. 6, April 15, 2007
DOI 10.1002/bit
resuspended to 3 mL. To determine if any cells lysed during
the reaction and subsequent washes, the amount of
hemoglobin in the supernatant was assayed with a UV–
visible spectrophotometer (Varian, Inc., Palo Alto, CA) as
described previously (Bonsen et al., 1977).
Partitioning
To determine the extent of bRBC modification with mPEG,
mPEG-bRBCs were partitioned in a two-phase system with
8,000 g/mol PEG and dextran (!71 kDa) (Dx) as described
previously (Bradley et al., 2002; Gavasane and Gaikar, 2003;
Lutwyche et al., 1995; Scott et al., 2000). The PEG stock
solution was prepared by mixing 30 g of 8,000 g/mol PEG
and 100 g of PEG solution buffer (0.15 mM NaCl, 6.84 mM
Na2HPO4, and 3.16 mM NaH2PO4, pH ¼ 7.4). The Dx
solution consisted of 30 g of Dx and 100 g of DI water. The
two-phase system was prepared as described previously
from the stock solutions with 5% (w/w) Dx, 4% (w/w) PEG
in 50 mL polypropylene tubes and equilibrated overnight.
The upper PEG-rich phase was drawn off, and the bottom
Dx-rich phase was collected by puncturing a hole at the
bottom of the tube. For the partitioning experiment, 12 mL
of mPEG-bRBCs was mixed with 750 mL of top PEG-rich
phase, followed by 750 mL of the bottom Dx-rich phase. The
mixture was inverted 20 times and allowed to equilibrate for
20 min. After the mixture of PEG, Dx, and cells was allowed
to equilibrate, mPEG-bRBCs remained in the PEG-rich
(top) phase and most of the unmodified, or slightly
modified, bRBCs settled to the PEG–Dx interface (Birkenmeier et al., 1994; Scott et al., 2000). Triplicate cell counts on
the top phase with a hematocytomer determined the
percentage of bRBCs remaining in the top phase, which
corresponded to the percentage of mPEG-bRBCs.
HPLC Analysis
The reaction supernatant was assayed on a BioSep-S2000
size exclusion HPLC column (Phenomenex, Torrance, CA)
to determine the amount of unreacted mPEG. The mPEG
buffer was used as the mobile phase. The mass of reacted
mPEG and extent of reaction was calculated from the
amount of unreacted PEG. Each sample of the original
mPEG solution was diluted to 1 mg mPEG/mL mPEG buffer
and 10–100 mL was injected to calibrate the peak area to
injected mass. To determine the remaining mPEG mass, the
reaction supernatant was diluted to approximately 1 mg
mPEG/mL mPEG buffer assuming half the initial mPEG
reacted. Twenty to 60 mL of the diluted reaction supernatant
was injected and the mass of mPEG was calculated from the
peak area.
Oxygen Binding Properties
As done previously in our group, a Hemox Analyzer (TCS
Scientific Corp., New Hope, PA) measured the oxygen
dissociation curves of unmodified bRBCs and mPEGbRBCs (Eike and Palmer, 2004; Gordon et al., 2005). Fifty
microliters of the bRBC suspension was mixed with 5 mL of
Hemox buffer, 10 mL of anti-foaming agent, and 20 mL of
Hemox A additive (TCS Scientific Corp). Air was bubbled
through the sample until it reached equilibrium at a pO2 of
!150 mmHg. As the sample was deoxygenated by the
addition of nitrogen, the Hemox Analyzer records the
fraction of oxygen saturated Hb as a function of pO2 until a
pO2 of !1.9 mmHg was reached. The Hemox Analyzer
software fitted the oxygen binding data to the Hill equation,
Equation 1, to yield the P50 and cooperativity of oxygen
binding (Hill coefficient) at physiological temperature
(378C). The data were also fit to a six-parameter step-wise
Adair equation used in later simulations (Eq. 2).
Y¼
Y¼
pOn2
n
þ P50
pOn2
A1 pO2 þ 2A2 pO2 þ 3A3 pO2 þ 4A4 pO2
4ð1 þ A1 pO2 þ A2 pO2 þ A3 pO2 þ A4 pO2 Þ
(1)
(2)
where Y ¼ fraction of oxygen saturated Hb, pO2 ¼ oxygen
partial pressure, P50 ¼ oxygen partial pressure when Hb is
half saturated with oxygen (Y ¼ 0.5), n ¼ cooperativity (Hill
coefficient), and Ai ¼ Adair parameters.
Rheology
A Carri-Med controlled stress rheometer with a 4 cm parallel
plate (Carri-Med Ltd., Dorking, Surrey, England, no longer
in manufacture) was used to measure the shear rate versus
applied shear stress (0 to !64 dyne/cm2) of mPEG-bRBCs at
40% hematocrit at 378C. Since blood is a non-Newtonian
fluid, the shear stress–shear rate curve was fit to the Casson
equation to regress the high shear rate viscosity and the yield
stress. Due to daily variances in setting the rheometer’s gap
width, the high shear rate viscosity was normalized such that
a control of plain washed bRBCs at 40% hematocrit in PBS
had a viscosity of 3 cp.
Capillary Wetting
The capillary wetting experiment examining the deformability of mPEG-bRBCs was described previously by Zhou
and Chang (2005) and Zhou et al. (2006). Briefly, mPEGbRBCs were diluted in PBS to 1% hematocrit. A 10-mL drop
was placed at the end of a glass capillary tube with a 21 mm
inner diameter (Polymicro Technologies, Phoenix, AZ). The
blood suspension enters the tube by capillary wetting
penetration. The meniscus flow was recorded using a highspeed video camera at 500 frames/s, and the meniscus
Gundersen and Palmer: PEGylated Bovine Erythrocytes
Biotechnology and Bioengineering. DOI 10.1002/bit
1201
velocity was calculated at five different lengths down the
capillary.
Oxygen Transport Simulations
To insure modified bRBCs were still capable of transporting
oxygen to tissues under a variety of simulation conditions,
oxygen transport simulations were conducted to model
oxygen transport from the mPEG-bRBCs in a capillary to
the surrounding tissue. Although the changes in oxygen
binding properties (P50 and n) were not very considerable,
simulations were conducted on all mPEG-bRBC solutions
and controls consisting of bRBCs and hRBCs.
The oxygen transport model described herein is a
modification of the Krogh tissue cylinder model, shown
in Figure 1. In this model, blood is assumed to behave as a
homogenous fluid with oxygen in equilibrium with
oxyhemoglobin as described mathematically by the specified
oxygen binding characteristics of the Adair equation. The
Hemox-derived Adair parameters were used for mPEGbRBC solutions and controls. The Adair parameters for
human RBCs were taken from McCarthy et al. (2001).
Oxygen diffuses through and out of the plasma and through
the capillary wall where some of it is consumed by the
capillary wall’s endothelial cells. Next, it diffuses through the
interstitial space to the tissue space where it is primarily
Figure 1. Modified Krogh tissue cylinder model including the capillary wall and
interstitial space between the capillary wall and the first tissue cells. [Color figure can
be seen in the online version of this article, available at www.interscience.wiley.com.]
consumed by the tissue cells. Tables I and II summarize the
dimensions and parameters used in the model.
Capillary Space
Differential mass balances on dissolved oxygen (O2) and
oxygenated hemoglobin (HbO2) were derived for the
capillary lumen. O2 and HbO2 were assumed to be in
chemical equilibrium, thus RO2 ;formation ¼ &RHbO2 ;formation .
Table I. Oxygen transport simulation parameters.
References
Capillary space
pO2,in
rc
L
Vz
[Hb]total
DO2;plasma
DHb,plasma
HO2;plasma
Adair Constants listed in Table II
Capillary wall
rw
rw- rc
DO2;wall
HO2;wall
Vmax,wall
Interstitial space
ri
ri- rw
DO2; interstitium
HO2;interstitium
Tissue space
rT
rT – ri
DO2;tissue
HO2;tissue
Km
Vmax
1202
20, 40, 60, 80, 95 mmHg
5 mm
0.1 cm
0.5 cm/s
8,800 mM
1.85E-05 cm2/s
Model parameters
Fournier (1999)
Fournier (1999)
Fournier (1999)
Fournier (1999)
Vadapalli et al. (2002)
5.30E-07 cm2/s
0.74 mmHg/mM
Vadapalli et al. (2002)
Fournier (1999)
Experimental and McCarthy et al. (2001)
5.3 mm
0.3 mm
8.73E-06 cm2/s
0.576 mmHg/mM
4.4615 mM/s
Vadapalli et al. (2002)
Vadapalli et al. (2002)
Vadapalli et al. (2002)
5.64 mm
0.34 mm
2.81E-05 cm2/s
0.798 mmHg/mM
Vadapalli
Vadapalli
Vadapalli
Vadapalli
13.7 mm
8.06 mm
6.30E-06 cm2/s
0.678 mmHg/mM
0.44 mmHg
26 (low glucose) mM/s
46 (high glucose) mM/s
Fournier (1999)
Vadapalli et al. (2002)
et
et
et
et
al.
al.
al.
al.
(2002)
(2002)
(2002)
(2002)
Vadapalli et al. (2002)
Vadapalli et al. (2002)
Fournier (1999)
Fournier (1999)
Fournier (1999)
Biotechnology and Bioengineering, Vol. 96, No. 6, April 15, 2007
DOI 10.1002/bit
Table II. Adair constants for mPEG-bRBCs and hRBCs.
4 mM
3 mM
2 mM
1 mM
0 mM
PBS
hRBCs
A1
A2
A3
A4
0.0441
0.0715
0.0349
0.0403
0.0585
0.0285
0.0153
3.917E-04
3.992E-04
6.550E-04
5.750E-04
3.768E-04
3.805E-04
1.100E-03
8.545E-06
3.851E-06
5.875E-13
5.601E-11
4.163E-13
3.868E-10
1.240E-07
4.331E-06
5.265E-06
3.388E-06
3.891E-06
2.998E-06
2.298E-06
1.810E-06
The two differential mass balances were combined to
eliminate the rate term yielding one equation in terms of
oxygen partial pressure (pO2) (Eq. 3). The equilibrium
relationship between HbO2 and O2 is described mathematically by the Adair equation. The simulated inlet pO2s
(z ¼ 0) (Eq. 4.a) are listed in Table I. The remaining
boundary conditions consisted of convective flux of oxygen
at the outlet of the capillary (z ¼ L), axial symmetry at the
center of the capillary (r ¼ 0), and continuity of oxygen at
the capillary wall (r ¼ rc) (Eqs. 4.b–d). The convective flux
boundary condition was used at z ¼ L in the capillary (and
all other regions), since it assumes that all endothelial and
tissue cells are absent and no more oxygen can be consumed
at that point. The length and radius of the capillary space
and plasma properties of blood are average representative
values for human capillaries as per Fournier (1999).
Vz ð1 þ mÞ
@pO2
@z
¼ ðDO2 ;plasma þ mDHbO2 ;plasma Þ
# 2
!
"$
@ pO2 1 @
@pO2
r
'
þ
r @r
@z 2
@r
(3)
@Y
where m ¼ HO2 ½Hb)total @pO
Y ¼ fraction of oxygen saturated
2
Hb
z¼0
r ¼ rc
pO2 ¼ pO2;in
(4.a)
z¼L
@pO2
¼0
@z
(4.b)
r¼0
@pO2
¼0
@r
(4.c)
DO2 ;plasma
@pO2
@pO2
¼ DO2 ;wall
@r
@r
cells. Equations 6.a–c represent the continuity boundary
conditions for the sides of the capillary wall, r ¼ rc and
r ¼ rw, and the isolated symmetry boundary condition for
both ends of the capillary at x ¼ 0 and x ¼ L. The oxygen
consumption kinetics of endothelial cells, diffusional
properties, and capillary wall dimensions were taken from
Vadapalli et al. (2002).
!
"#
!
"$
DO2 ;plasma @2 pO2 1 @
@pO2
r
0¼
þ
r @r
HO2 ;wall
@z 2
@r
þ Vmax;wall
(5)
z ¼ 0 and z ¼ L
r ¼ rc
r ¼ rw
(6.a)
@pO2
@pO2
¼ DO2 ;wall
@r
@r
(6.b)
@pO2
@pO2
¼ DO2 ;interstitium
@r
@r
(6.c)
DO2 ;plasma
DO2 ;wall
@pO2
¼0
@r
Interstitial Space
The interstitial space was modeled with a differential mass
balance on O2 with model dimensions as per Vadapalli et al.
(2002) (Eq. 7). It is assumed that the interstitial space has no
tissue cells, thus oxygen is not produced or consumed in this
region. The boundary conditions consisted of continuity for
both sides of the region, r ¼ rw and r ¼ ri, and isolated
symmetry for both ends of the interstitial space at x ¼ 0 and
x ¼ L (Eqs. 8.a–c).
0¼
!
DO2 ;interstitium
HO2 ;interstitium
"#
!
"$
@2 pO2 1 @
@pO2
r
þ
r @r
@z 2
@r
z ¼ 0 and z ¼ L
@pO2
¼0
@r
(7)
(8.a)
@pO2
@pO2
¼ DO2 ;interstitium
@r
@r
(8.b)
@pO2
@pO2
¼ DO2 ;tissue
@r
@r
(8.c)
r ¼ rw
DO2 ;wall
r ¼ ri
DO2 ;interstitium
(4.d)
Tissue Space
Capillary Wall
A differential mass balance on O2 was derived for the
capillary wall, since it is assumed that Hb and HbO2 cannot
diffuse through the wall (Eq. 5). Zeroth order kinetics
describe O2 consumption by the capillary wall endothelial
Equation 9 shows the differential O2 mass balance in the
tissue space with first-order Michaelis–Menten kinetics
describing oxygen consumption of pancreas islet tissues at
high and low glucose levels. The two glucose levels
correspond to high and low Vmax, respectively, to provide
a range of oxygen consumption rates. The boundary
Gundersen and Palmer: PEGylated Bovine Erythrocytes
Biotechnology and Bioengineering. DOI 10.1002/bit
1203
conditions consisted of isolated symmetry for the three sides
of the modeled tissue space at x ¼ 0, x ¼ L, and r ¼ RT, and a
continuity boundary condition between the interstitium and
tissue space (Eqs. 10.a–b). The oxygen consumption kinetics
and tissue dimensions were modeled as per Fournier (1999).
0¼
!
DO2 ;tissue
HO2 ;tissue
þ
"#
Vmax pO2
Km þ pO2
!
"$
@2 pO2 1 @
@pO2
r
þ
r @r
@z 2
@r
(9)
z ¼ 0; z ¼ L; and r ¼ RT
r ¼ ri
DO2 ;interstitium
@pO2
¼0
@r
@pO2
@pO2
¼ DO2 ;tissue
@r
@r
(10.a)
(10.b)
Results
Partitioning experiments suggest that bRBCs are being
effectively surface conjugated with mPEG. bRBCs, initially
at 12% hematocrit in PBS reacted with 0–4 mM SPA-mPEG.
Figure 2 shows the partitioning results as a ratio of the
percentage of mPEG-bRBCs in the top PEG-rich phase to
the percentage of unreacted (PBS control) bRBCs in the
PEG-rich phase. The 0-mM control corresponding to that
day’s experiment overlaps the experimental sample. Since
there was some variability in the amount of partitioning
between experiments done on different days, all data were
normalized to the PBS control to be able to compare the
data. bRBCs modified with 2, 3, and 4 mM SPA-mPEG
noticeably partitioned into the top (PEG-rich) phase to a
greater extent versus the controls. Reactions with 4 mM
mPEG partitioned up to 10 times more compared to the PBS
control. bRBCs reacted with 2 and 3 mM mPEG partitioned
about two and six times more compared to the control,
respectively. The reaction with 1 mM mPEG had only
slightly increased partitioning. As would be expected, greater
concentrations of mPEG resulted in the formation of more
mPEG-bRBCs favoring the PEG phase, thus indicating a
higher amount of mPEG surface coverage.
HPLC analysis (Fig. 3) shows the amount of unreacted
mPEG remaining in solution after the 1-h reaction period.
The percentage of reacted SPA-mPEG was greatest with
1 mM SPA-mPEG at 50.5% with other mPEG concentrations (2, 3, and 4 mM) resulted in approximately 38% of
reacted mPEG. At 4 mM mPEG, 90.3 mg mPEG reacted with
bRBCs. At lower mPEG concentrations, 29.8, 44.2, and
66.4 mg of mPEG reacted at 1, 2, and 3 mM of mPEG,
respectively. Since bRBCs reacted at the same hematocrit,
this corresponds to more mPEG molecules being conjugated
per cell at higher initial mPEG concentrations. Assuming
bRBCs have a diameter of about 6 mm, these results
correspond to 1.4, 2.1, 3.2, and 4.3 ' 108 mPEG molecules/
bRBC for initial concentrations of 1, 2, 3, and 4 mM mPEG,
respectively.
Oxygen binding measurements show a slight drop in P50
with increased bRBC surface coverage with mPEG. The
oxygen dissociation curves were fitted to the Hill Equation
to regress the P50 and cooperativity (Hill coefficient), shown
in Figure 4A and B (Voet and Voet, 1995). The mPEG layer
conjugated to the surface of bRBCs slightly increases the
overall O2 affinity as shown by the left shifted curves
Figure 2.
Percentage of mPEG-bRBCs remaining in the top, PEG-rich phase
after partitioning, normalized to the corresponding unreacted bRBCs (PBS control) in
the PEG-rich phase. bRBCs initially at 12% hematocrit were reacted with the specified
concentrations of SPA-mPEG. After reaction, bRBCs were reconstituted in a PEG–Dx
solution. bRBCs modified with mPEG should remain in the top PEG-rich phase. Results,
in solid bars, show the percentage of mPEG-bRBCs remaining in the PEG-rich phase/
percentage of bRBCs that reacted with PBS in the PEG phase after the partitioning
experiment. Overlapping, in dotted bars, is the additional control of 0 mM mPEG
corresponding to that sample. Error bars represent the standard error in cell counts
from three samples propagated through to the normalized percentage. [Color figure
can be seen in the online version of this article, available at www.interscience.
wiley.com.]
1204
Figure 3. Mass of SPA-mPEG that reacted with bRBCs (solid bars) and the
percentage of reacted mPEG (dotted bars) derived from HPLC measurements. Left
axis: mass of mPEG reacted; right axis: percent of initial mPEG that reacted with
bRBCs. Error bars represent the standard error in HPLC peak area averaged from three
experiments propagated through to the percentage of reacted mPEG. [Color figure can
be seen in the online version of this article, available at www.interscience.wiley.com.]
Biotechnology and Bioengineering, Vol. 96, No. 6, April 15, 2007
DOI 10.1002/bit
Figure 5. High shear rate viscosities of mPEG-bRBCs and controls normalized to
3 cp for that day’s 40% hematocrit control. bRBCs were reacted with 4–0 mM mPEG or
unreacted PBS as indicated and resuspended to 40% hematocrit (*1.5% hematocrit).
The dotted line is at 3 cp, which is the average high shear rates viscosity for human
blood at 40% hematocrit with or without fibrinogen. Error bars represent the standard
error of the average fitted high shear rate viscosity of three experiments.
Figure 4. A: P50 and B: cooperativity (Hill coefficient) of mPEG-bRBCs as
determined from curve fitting experimental oxygen dissociation curves to the Hill
equation. The dotted line in (A) shows the average P50 for human RBCs !27 mmHg. All
samples were mixed with equal volumes of bRBCs at 12% hematocrit and the
corresponding SPA-mPEG concentration. Error bars represent the standard error
of the average of three experiments.
corresponding to a lower P50. The average P50 for bRBCs is
27.92 mmHg, which drops to 26.04 mmHg for bRBCs
reacted with 2 mM mPEG, and finally to 24.38 mmHg for a
reaction with 4 mM mPEG. The cooperativity of Hb’s O2
binding (Fig. 4B) is maintained with PEGylation of the
bRBC membrane yielding Hill coefficients >2.
The viscosity results in Figure 5 show similar high shear
rate viscosities for modified bRBCs and the control group of
!3 cp. All samples were resuspended to a hematocrit of
40 * 1.5%. Some of the variability in the measured viscosity
is due to the slightly varying hematocrits. bRBCs reacted at
the highest concentration, 4 mM mPEG, exhibited a
measured viscosity of 2.8 cp. The remaining samples reacted
at 0, 1, 2, 3 mM mPEG, and PBS exhibited apparent
viscosities of 2.9, 2.6, 2.7, 2.3, 3.1 cp, respectively. Although
the rheometer software fitted the experimentally measured
shear stress versus shear rate to the Casson equation for
blood, the yield stress values were all very low, approximately zero, and out of the measurement range of the
instrument and will not be included.
After reacting bRBCs with mPEG, mPEG-bRBCs were
washed six times in PBS. After each centrifugation step, the
supernatant was collected and tested for free Hb to
determine if the conjugation reaction with mPEG or the
subsequent PBS washes elicited cell lysis (data not shown).
On average, reactions with up to 4 mM mPEG had less than
1% of acellular Hb in the supernatant for each wash.
Therefore, the reaction alone does not immediately
compromise the bRBC’s membrane integrity.
Capillary wetting experiments showed some unexpected
results. The meniscus velocity remained the same for all
mPEG-bRBCs, as shown in Figure 6. Modified bRBCs
displayed similar meniscus packing behavior compared to
normal bRBCs. However, mPEG-bRBCs would pack into
periodic plugs. Figure 7 shows sequential screen shots of the
Figure 6. Meniscus velocity versus the wetted capillary length of several mPEG
modifications of bRBCs. [Color figure can be seen in the online version of this article,
available at www.interscience.wiley.com.]
Gundersen and Palmer: PEGylated Bovine Erythrocytes
Biotechnology and Bioengineering. DOI 10.1002/bit
1205
Figure 7. Capillary wetting snapshots. A: Unmodified bRBCs in PBS pack at the meniscus (left pane 1) and will migrate to the axial centerline further downstream in panes 2
and 3. B: Modified cells (at 4 mM mPEG) will still pack at the meniscus (left pane 1) and migrate to the axial centerline (pane 2). However, the amount of cells at the axial centerline
will slowly decline until the tube is empty (pane 3) and then be followed by a second slug of meniscus-like packed cells (pane 4). [Color figure can be seen in the online version of this
article, available at www.interscience.wiley.com.]
video of (A) unreacted bRBCs and (B) 4 mM mPEG
modified mPEG-bRBCs as they travel down the length of the
glass capillary. The left hand frame (Frame 1) is the first
snapshot of the meniscus. Frames 2–4 show subsequent
snapshots in the order they were taken. As can be seen in
Figure 7A, normal bRBCs will pack at the meniscus, Frame
1, and further upstream are migrating to the capillary
centerline, Frames 2 and 3. PEGylated bRBCs also pack at
the meniscus, as seen in Figure 7B Frame 1, and upstream of
the meniscus will migrate to the center, Frame 2. However,
no to very little bRBCs are present further upstream, Frame
3, until a second plug of cells appears in Frame 4. This
second plug of cells follows a similar trend of packing at the
beginning of the plug (as opposed to the meniscus) and
migrating to the centerline after the plug.
For oxygen transport simulations, examination of the
axial centerline (r ¼ 0) pO2 profile for all mPEG-bRBCs
(Fig. 8A) shows that the oxygen concentration decreases
Figure 8. Simulated oxygen concentration profiles at the axial centerline (r ¼ 0) at (A) pO2,in ¼ 95 mmHg and (B) pO2,in ¼ 20 mmHg. Simulated oxygen concentration profiles at
the tissue radius (r ¼ RT) at (C) pO2,in ¼ 95 mmHg and (D) pO2,in ¼ 20 mmHg. The solid lines represent simulations at low glucose levels, low Vmax, and the dotted lines represent
simulations at high glucose levels, high Vmax. [Color figure can be seen in the online version of this article, available at www.interscience.wiley.com.]
1206
Biotechnology and Bioengineering, Vol. 96, No. 6, April 15, 2007
DOI 10.1002/bit
down the capillary centerline as the pancreatic tissue and
endothelial cells consume oxygen. The pO2 profile at the
tissue edge (Fig. 8B) also exhibits the same decreasing trend
for various pO2,in and Vmax values, but shifted to lower pO2s
due to its increased distance from the oxygen source in the
capillary. In the extreme case of a high Vmax value and
hypoxia (pO2,in ¼ 20 mmHg), the tissue pO2 has a lower
limit of 15.5 mmHg.
The percentage of total incoming oxygen transferred to
the tissues was determined from an overall oxygen mass
balance on the capillary space. Figure 9A displays the percent
decrease of total dissolved oxygen in the capillary for
modified bRBCs at various inlet pO2s at low Vmax. Since the
oxygen consumption rate is dependent on the pO2 via
Michaelis–Menten kinetics, it is expected that more O2 is
consumed at higher pO2s. However, since more oxygen is
available at higher inlet pO2s, the percentage of O2
consumed is lower at higher initial pO2s. Equivalently,
there is a larger percent decrease in O2 at low inlet pO2s, even
though the tissues consume less oxygen.
Furthermore, as would be expected, the larger percentage
of oxygen lost to tissues at a high Vmax value at a given
entering pO2 is due to the higher tissue oxygen consumption
rate as the cells metabolize more oxygen (Fig. 9B). Although,
the P50 generally decreased with increasing initial mPEG
concentration, the Hill coefficient did not follow this trend.
This would correspond to varying Adair constants, causing a
lack of an apparent trend between mPEG concentration and
the percentage of oxygen transported to the tissue space.
Discussion
Figure 9. Percentage of total incoming oxygen lost from the capillary to the
tissues at (A) low glucose levels, low Vmax, and (B) high glucose levels, high Vmax.
C: Representative %O2 transfer to tissue values for both high Vmax (dotted lines) and
low Vmax (solid lines). [Color figure can be seen in the online version of this article,
available at www.interscience.wiley.com.]
As the primary hRBC donor population shrinks in size, a
viable substitute for hRBCs is in high demand. Although
modified Hbs show good potential as oxygen carriers, they
still have many problems to overcome. Also, more
importantly the production of modified Hbs and mPEGhRBCs are highly dependent on the dwindling supply of
human blood. Hence, modified bRBCs constitute a possible
solution to ease the demand on human blood donations and
the cost of donated blood. Although the amount of mPEG
required to immunologically silent hRBCs should be
approximately the same as for bRBCs, the main cost savings
would derive from the blood source (!$100 less for bRBCs).
The evaluation and control of select physical properties of
mPEG-bRBCs is essential for the successful application of
this novel xenotransfusion technology. For example, if the
mPEG-bRBCs cannot support normal oxygen delivery or
are too rigid, they could potentially clog the capillaries or
severely impair oxygen transport to surrounding tissues.
Hence, this study focused on elucidating the physical
properties of mPEG-bRBCs, that is, oxygen binding,
rheology, and deformability.
Since the primary reason for mPEG modification is to
effectively cover antigenic sites on the bRBC membrane, it is
important to determine how well bRBCs are surface
conjugated with mPEG. Both partitioning and HPLC
analysis (Figs. 2 and 3) showed that bRBCs were successfully
modified with SPA-mPEG in a dose-dependent manner.
Although the percentage of initial mPEG reacting with
bRBCs levels off, the mass of SPA-mPEG reacting with
bRBCs increases with increasing mPEG concentration up to
4 mM. The results in Figures 2 and 3 agree, showing that
more mPEG molecules are conjugated to the bRBC surface
when reacted at higher initial mPEG concentrations. These
results are in agreement with Bradley et al. (2001, 2002), who
showed that surface coverage of mPEGs on hRBCs increased
with increasing initial mPEG concentrations. The surface
coverage of mPEG on the bRBC membrane should increase
Gundersen and Palmer: PEGylated Bovine Erythrocytes
Biotechnology and Bioengineering. DOI 10.1002/bit
1207
until it reaches a maximum saturation value such that
increasing the initial mPEG concentration will not affect the
amount of surface conjugated mPEG molecules. This will be
influenced by the number of possible cross-linking sites
available on the bRBC surface and steric exclusion between
neighboring mPEG molecules. Since the reactive ester group
targets positively charged amine groups, it should not react
with itself at high concentrations (Nektar, 2006). Even
though the average number of mPEG molecules per cell was
quite high, some bRBCs remained unmodified or slightly
modified due to possible poor mixing or were trapped near
the glass wall of the reaction vessel during the reaction. In
this study, partitioning was used only as a qualitative test to
determine the relative degree of mPEG modification. By
changing the molecular weight and concentrations of the
PEG and Dx partitioning solutions, this technique can be
‘‘fine-tuned’’ so all unmodified and lightly modified cells
will settle at the PEG–Dx interface, while successfully
modified bRBCs will remain in the PEG-rich layer. This will
be especially important for potential clinical application as
the mPEG-bRBC solution will have to be free of unmodified
and poorly modified bRBCs to not elicit an immune
response.
There was minimal acellular Hb present in the reaction
mixture after the 1-h reaction period and in the supernatant
during subsequent PBS washes. Less than 5% of cellular Hb
was found in the supernatant after the five washes. This
small amount could be enough to initiate an immune
response if left in the solution. However, with washes and
resuspension in the appropriate media for storage, such as
citrate phosphate dextrose for hRBCs, this should be able to
be removed before transfusion.
Modified bRBC solutions must be able to be resuspended
to a hematocrit exhibiting physiological and rheological
properties, while maintaining efficient oxygen delivery. In
normal human blood flow, the shear rate is !100 s&1 where
blood behaves like a Newtonian fluid with a viscosity of 3 cp
(Fournier, 1999). Our rheometry studies showed that
mPEG-bRBCs at 40 * 1.5% hematocrit exhibited a high
shear rate viscosity of !3cp. The appearance of a yield stress
and non-Newtonian behavior of normal whole blood at low
shear rates is due to RBC aggregate formation and other
plasma proteins, particularly fibrinogen (Armstrong et al.,
1997; Fournier, 1999). Thus, the low yield stress values
observed in our experiments are expected for RBCs
resuspended in PBS due to the absence of fibrinogen and
other plasma contents (Fournier, 1999). These results are in
agreement with the general behavior of blood solutions and
with experiments by Armstrong et al. (1997) who showed
that PEGylation of hRBCs resulted in lower viscosities at low
shear rates (lower yield stress values) due to conjugated
mPEG hindering RBC aggregate formation. There is a slight
decrease in viscosity for bRBCs reacted at 2 and 3 mM
mPEG. However, this is most likely due to the slight
variances in the hematocrit than a function of PEGylation.
It is also important for mPEG-bRBCs to be flexible, as
RBCs have to deform to fit through smaller sized capillaries.
1208
Even though bRBCs are slightly smaller than hRBCs, human
capillaries can possess a diameter less than bRBCs as well. In
capillary flow, shear-induced migration will cause deformable particles to migrate to the axial centerline, while
perfectly rigid particles will remain uniformly suspended
(King and Leighton, 2001). Since the deformable particles at
the centerline travel faster than the average velocity, they will
begin to pack at the meniscus of the flow. This will form a
plug at the meniscus and will subsequently cause the
meniscus to slow down. Thus, more deformable particles
will lead to slower capillary penetration. The capillary
wetting experiments showed mPEG-bRBCs to be deformable, even though they exhibited slightly different entrance
phenomena in a glass capillary compared to normal bRBCs.
This should not affect in vivo properties, since this particular
glass capillary entrance geometry is not observed in vivo.
Since capillary flow was the only concern, the most
important outcome of this experiment is that the mPEGbRBCs still packed at the meniscus and migrated to the axial
centerline. In accordance with results from Zhou and Chang
(2005), mPEG-bRBCs can be considered deformable
particles. The reason for the different entrance behaviors
between mPEG-bRBCs and normal bRBCs could be due to
mPEG molecules preventing cell–cell interactions or
differences in surface charge density. However, future zeta
potential measurements will be needed to confirm this
hypothesis.
Naturally it is essential for mPEG-bRBCs to perform
RBC’s most important function of delivering oxygen to the
tissues. Hence, the oxygen binding properties of mPEGbRBCs should be similar to hRBCs. Although the P50
dropped slightly, mPEG-bRBCs maintained a high P50 for
bRBCs which reacted at lower mPEG concentrations close to
the hRBC physiological value of 27 mmHg. Modified bRBCs
maintained cooperative oxygen binding with cooperativities
>2 as would be expected, since mPEG modification of the
cell’s surface should not have an affect on the cooperativity
of Hb. Scott and Chen (2004) reported similar drops in P50
with mPEG modifications up to 5 mM cyanuric chlorideactivated mPEG on human RBCs with no change in
cooperativity. The reason for the slight drop in P50 is still
under investigation and poorly understood. However, it is
assumed this change is not due to the mPEG diffusing inside
the cell and reacting with Hb directly. Methoxy PEG should
not be able to enter the cell due to its hydrophilic nature.
Thus, it is highly unlikely that SPA-mPEG reacts directly
with Hb to influence Hb-O2 binding.
Next, the experimentally measured oxygen binding
parameters of mPEG-bRBCs were used as input variables
in oxygen transport simulations to confirm that the slight
decrease in P50 of mPEG-bRBCs can still result in the
delivery of sufficient oxygen to tissues similarly to hRBCs
even under extreme oxygenation. The most important
outcome from these simulations shows mPEG-bRBCs
ability to maintain the tissue pO2. Since normal, nontumorgenic tissues require a minimum pO2 of 7–10 mmHg
to avoid hypoxia, the mPEG-bRBCs must be able to
Biotechnology and Bioengineering, Vol. 96, No. 6, April 15, 2007
DOI 10.1002/bit
transport enough oxygen to sustain tissue pO2s above this
limit (Harrison and Blackwell, 2004; Vaupel, 2004). As seen
from the oxygen transport simulations, the tissue pO2 level
remained above 10 mmHg for all surface coverages of mPEG
on the bRBC surface even under extreme oxygenation
scenarios characterized by low entering pO2s and high
oxygen consumption rates in the tissues. These simulations
demonstrate that mPEG-bRBCs should be able to transport
the necessary amount of oxygen to tissues under normal
physiological conditions.
Figure 8 does not appear to agree with the convective flux
boundary condition imposed at z ¼ L. The O2 profile is
expected to decrease to a slope of zero at the end of the
capillary due to the exiting boundary condition. This was
not observed since there are pancreatic and endothelial cells
consuming oxygen until z ¼ L. Therefore, the pO2 will
continue to decrease until either there is no more O2
consuming tissue, which is located at z ¼ L in this
simulation, or the oxygen is completely consumed in the
capillary and pO2 ¼ 0.
Should bRBCs prove to be a feasible hRBC substitute, they
could help reduce the demand on human blood transfusions. Also, mPEG-bRBCs could be cross-linked with
gluteraldehyde to further alter the P50 for oxygenation in
bio-artificial devices (Gordon et al., 2005; Sullivan et al.,
2006). Before application of this novel blood substitute, the
most important concern will be the safety of the bovine
blood supply. In addition to Quad Five’s quarantine and
screening procedures, the presence of TSE in cow’s blood
can be excluded by testing two generations of founders and
quarantining the offspring (Johnstone et al., 2004).
Furthermore, it will be necessary to obtain a continuous
fresh bovine blood source for further tests, especially for
clinical trials and Food and Drug Administration approval,
however, this is beyond the scope of this current study.
Conclusions
The results of this study show that bRBCs can be effectively
surface conjugated with mPEG, just as hRBCs can, without
compromising the cell’s structure and oxygen binding
functions. There is significantly more partitioning of mPEGbRBCs in the PEG-rich phase compared to control bRBCs.
HPLC analysis also shows that bRBCs are being conjugated
with SPA-mPEG due to the significant decrease in unreacted
mPEG molecules. Also, mPEG-bRBCs demonstrated similar
viscosities compared to unmodified bRBCs and hRBCs.
Modified bRBCs retain their ability to bind and release
oxygen, as shown by only moderate changes in oxygen
binding properties, such as the P50 and cooperativity
coefficient. This was further confirmed in oxygen transport
simulations, which demonstrated that PEGylation does not
affect the ability of mPEG-bRBCs to deliver oxygen to tissues
under a variety of oxygenation conditions. Even under the
most extreme circumstance of pO2,in ¼ 20 mmHg and high
Vmax value, tissues still received enough oxygen to remain
viable (>10 mmHg). Taken together, these results demonstrate that mPEG-bRBCs at any level of PEGylation can
function as a suitable human RBC substitute from a physical
standpoint. Of course, future immunological studies are
needed to prove the feasibility of using mPEG-bRBCs as a
hRBC substitute.
This research was supported by United States Public Health Service
grants HL078840 and DK070862 to A.F.P.
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