0022-3565/99/2882-0665$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
JPET 288:665–670, 1999
Vol. 288, No. 2
Printed in U.S.A.
Polyethylene Glycol-Modified Liposome-Encapsulated
Hemoglobin: A Long Circulating Red Cell Substitute1
W. T. PHILLIPS, R. W. KLIPPER, V. D. AWASTHI, A. S. RUDOLPH, R. CLIFF, V. KWASIBORSKI and B. A. GOINS
Radiology Department, University of Texas Health Science Center at San Antonio, San Antonio, Texas (W.T.P., R.W.K., V.D.A., B.A.G.); and
Center for BioMolecular Science and Engineering, Naval Research Laboratory, Washington, DC (A.S.R., R.C., V.K.)
Accepted for publication September 9, 1998
This paper is available online at http://www.jpet.org
Red cell substitutes are currently under development as
resuscitative agents for use in trauma and surgery associated
with high blood loss. Research efforts to develop an adequate
red cell substitute have focused primarily on oxygen-carrying
formulations based on either perfluorochemicals or hemoglobin (Winslow, 1992; Spence, 1995). An important consideration related to the clinical use of red cell substitutes is how
long they remain in circulation. An adequate circulation time
for a red cell substitute enables it to deliver a physiologically
significant amount of oxygen to the patient’s tissues.
For such reasons, increasing the circulation persistence of
a red cell substitute, and therefore prolonging its oxygen
delivery, has been a long-standing objective for researchers
in this field. Various strategies have been implemented to
meet this objective. For example, intramolecular and intermolecular crosslinking of hemoglobin has increased the circulation half-life of hemoglobin from 0.5 to 1.5 h for unmodified hemoglobin to 6 to 12 h (Hess et al., 1989). Another
technique that has been used to increase the circulation
persistence of hemoglobin is conjugation with polyethylene
Received for publication March 5, 1998.
1
This work was supported by National Institutes of Health Grant RO1HL53052, US Naval Medical Research and Development Command, and US
Army Medical Research and Development Command (for supplying hemoglobin).
distribution data at 48 h revealed that 51.3 6 3.4% of the
technetium-99m-PEG-LEH remained in circulation, a greater
than 3-fold increase in the circulation half-life compared with
circulation half-lives previously reported for non-PEG-containing LEH formulations. The liver had the greatest accumulation
at 48 h (12.7 6 0.7%), followed by bone marrow (6.2 6 0.1%),
whereas the spleen had only 1.4 6 0.2%. The addition of
PEG-PE to the LEH formulation greatly prolongs the circulation
persistence of LEH and represents a significant step in the
development of red cell substitutes with prolonged oxygen
delivery.
glycol (PEG) (Smyth et al., 1947; Nucci et al., 1991; Katre,
1993). PEG is a biologically inert compound regularly used in
both cosmetic and pharmaceutical preparations and is considered safe for use by the Food and Drug Administration
(Smyth et al., 1947; Lasic, 1996). In a rabbit model, conjugation of hemoglobin with PEG extended its circulation half-life
to 43 h (Conover et al., 1997).
The encapsulation of hemoglobin in liposomes is yet another approach that results in prolonged circulation persistence of the hemoglobin and, at the same time, shifts its
clearance route from the kidneys to the reticuloendothelial
system (RES) (Chang, 1957; Farmer and Gaber, 1987; Rudolph et al., 1991; Tsuchida, 1994; Sakai et al., 1997).
Surface modification of the liposome-encapsulated hemoglobin (LEH) with PEG is a potential additional strategy to
further increase the circulation persistence of LEH. PEGsurface modification has been shown to increase the circulation persistence of liposomes by decreasing recognition and
uptake (Allen et al., 1991; Torchilin et al., 1992, 1994). Most
previous studies have shown that PEG incorporation greatly
increases the circulation persistence of liposomes and allows
for greater drug delivery to tumors (Gabizon et al., 1990).
Recently, the value of PEG-liposome surface modification for
increasing drug delivery has been questioned in situations
where large liposome lipid doses are administered (Parr et
ABBREVIATIONS: LEH, liposome-encapsulated hemoglobin; PEG, polyethylene glycol; DSPE, distearoyl phosphoethanolamine; 99mTc, technetium-99m; HMPAO, hexamethylpropyleneamine oxime; % ID, percentage of injected dose; RES, reticuloendothelial system; PEG-LEH, polyethylene glycol-coated liposome-encapsulated hemoglobin; DMPG, dimyristoyl phosphatidylglycerol.
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ABSTRACT
A major obstacle in the development of red cell substitutes has
been overcoming their short circulation persistence. In this
study, distearoyl phosphoethanolamine polyethylene glycol
5000 (PEG-PE) (10 mol%) was added to the formulation of
liposome-encapsulated hemoglobin (LEH) to decrease reticuloendothelial system uptake and prolong LEH circulation persistence. PEG-LEH was radiolabeled with technetium-99m, infused into rabbits (25% of blood pool at 1 ml/min) (n 5 5), and
monitored by scintigraphic imaging at various times out to 48 h.
At 48 h, animals were sacrificed, and tissue samples were
collected for counting in a scintillation well counter. Tissue
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Phillips et al.
Vol. 288
Materials and Methods
PEG-LEH Preparation. PEG-LEH was produced using sterile
technique by microfluidization after rehydration of a dried lipid film
of distearoyl phosphatidylcholine (DSPC), cholesterol, distearoyl
phosphoethanolamine-N-(polyethylene glycol 5000) (DSPE-PEG),
and a-tocopherol (mole ratio 50:38:10:2) with aa-crosslinked hemoglobin (Bionetics, Rockville, MD) containing 30 mM reduced glutathione (Sigma Chemical Co., St. Louis, MO), 10 mg/ml human serum
albumin, and 9% sucrose. After production, PEG-LEH was separated
from unencapsulated hemoglobin by ultrafiltration through a 300kDa polysulfone filter. The final PEG-LEH preparation was tested as
previously described (Farmer and Gaber, 1987; Goins et al., 1994),
and the characteristics of PEG-LEH are shown in Table 1.
PEG-LEH Labeling. PEG-LEH was labeled using a previously
described and validated method for labeling liposomes with 99mTc
(Rudolph et al., 1991; Phillips et al., 1992; Goins et al., 1993; Phillips
and Goins, 1995). PEG-LEH (4 ml) was incubated for 30 min with 2
ml of 99mTc-hexamethylpropyleneamine oxime (HMPAO) (Ceretec;
Amersham, Arlington Heights, IL) that had been previously incubated for 5 min with 10 mCi of 99mTc-sodium pertechnetate in 5 ml
of 0.9% saline. After the labeling process, the liposomes were separated from free 99mTc by passage over a Sephadex G-25 column.
Labeling efficiency was calculated by comparing precolumn and postcolumn values using a dose calibrator (Radex Model Mark J, Houston, TX). Labeling efficiency ranged from 57 to 65%.
Animal Experiments. Animal experiments were performed under the National Institutes of Health Animal Use and Care guidelines and were approved by the University of Texas Health Science
Results
TABLE 1
Characteristics of PEG-LEH
Unimodal size distribution
Phospholipid content
Hemoglobin content
Endotoxin level
Sterility
Center at San Antonio Institutional Animal Care Committee. Male
New Zealand White rabbits (2.5–3.0 kg), that had been fasted the
night before the study, were anesthetized with 50 mg of ketamine-10
mg of xylazine/kg b.wt. i.m. One ear of the rabbit was catheterized
with a 23-gauge venous line, and the other ear was catheterized with
a 20-gauge arterial line. Blood samples were drawn from the arterial
line, and 99mTc-PEG-LEH was infused in the venous line. Rabbits
were then placed in the supine position under a Picker (Cleveland,
OH) large-field-of-view gamma camera interfaced with a Pinnacle
imaging computer (Medasys, Ann Arbor, MI), and image acquisition
was begun with a low-energy all-purpose collimator as the 99mTcPEG-LEH solution was introduced at 1.0 ml/min. The animals received a total dose of 99mTc-PEG-LEH (3–5 mCi 99mTc activity, 36.9
ml, 1.13 g phospholipid/kg b.wt., 0.16 g hemoglobin/kg b.wt.) equivalent to 25% of their circulating blood volume based on 59 ml/kg b.wt.
(Kaplan and Timmons, 1979). One-minute dynamic 64 3 64 pixel
scintigraphic images were acquired over a continuous period of 2.5 h.
Blood was drawn into capillary tubes (50 ml) at various times after
99m
Tc-PEG-LEH injection to monitor circulation persistence. At both
24 and 48 h postinfusion, static images and blood samples were
acquired. After 48 h, the animals were euthanized with an overdose
of pentobarbital. Tissue samples were collected, weighed, and
counted for radioactivity in a scintillation well counter (Canberra,
Meridan, CT) for calculation of biodistribution. Calculations used to
estimate the total percent of the injected dose (% ID) in blood,
muscle, skin, and bone marrow were blood volume, 5.9 ml/100 g
b.wt.; muscle, 45 g/100 g b.wt.; and skin, 10 g/100 g b.wt., respectively. The % ID in the bone marrow was estimated to be 12 times the
% ID in one femur (Dietz, 1944).
Control studies with 99mTc-labeled red blood cells were performed
for the blood pool contribution subtraction as follows. Briefly, heparinized blood (6.0 ml) was withdrawn from rabbits (n 5 3) and added
to a red blood cell labeling kit (Ultratag; Mallinckrodt, St. Louis,
MO). After labeling with 99mTc, the blood was reinfused into each
rabbit through an ear vein. After adequate equilibration of the
99m
Tc-labeled red blood cells (20 min), scintigraphic images were
acquired and analyzed to determine the percent distribution of total
acquired counts in the heart, liver, and spleen.
Image Analysis. Image analysis was performed using a nuclear
medicine analysis workstation (Pinnacle computer; Medasys, Ann
Arbor, MI). Whole body images were decay corrected, and then
regions of interest were drawn over the liver, spleen, and heart in
images acquired at various times after the infusion of 99mTc-PEGLEH. A box was drawn around the whole animal in each image at the
same time points to represent the counts from the total injected dose
given during the study. The counts in each organ were converted to
a percentage of total body counts. The distribution percentages in a
given organ for each animal were averaged, and standard errors
were calculated. Images were corrected for blood pool contribution
using a previously described technique (Rudolph et al., 1991).
Blood Clearance Analysis. Blood samples were counted for
radioactivity in a scintillation well counter. The radioactive counts
were decay corrected to account for the half-life of 99mTc and plotted
against the sampling time to generate blood clearance curves. Blood
clearance curves were fitted to a two-compartment model to generate
circulation half-lives using Scientist for Windows software with supplemental Pharmacokinetic Model Library (MicroMath, Salt Lake
City, UT).
193 nm
110.5 mM
1.2 g/dl
18 EU/ml
No growth for 14 days in thioglycollate
media at 37°C
An image of a rabbit acquired 2 h after the infusion of
Tc-PEG-LEH is shown in Fig. 1A. The distribution of
99m
Tc activity visualized at this time is due to 99mTc activity
in the heart and other vasculature. The 99mTc activity in the
liver is primarily due to the vascularity of the liver, and no
99m
Tc activity can be detected in the spleen. This distribution
99m
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al., 1997). With large doses of lipid such as that given when
LEH is administered, the RES could potentially become saturated so that PEG surface modification may provide only
minimal extension of the circulation half-life compared with
standard liposomes.
Another important factor controlling circulation half-life of
a liposome-encapsulated agent is the precise mole percentage
(mol%) of polyethylene glycol distearoyl phosphoethanolamine (PEG-DSPE) included in the liposome formulation.
The mol% and the molecular weight of the PEG headgroup
appear to be critical for successfully increasing the circulation persistence of LEH. Previous attempts to significantly
prolong the circulation of LEH with 5 mol% PEG-PE were
unsuccessful (Zheng et al., 1994). The present study was
undertaken to determine whether the addition of a higher 10
mol% PEG-DSPE with a PEG headgroup of greater molecular weight (5000 versus 1900) would increase the circulation
persistence and change the tissue biodistribution of LEH. To
monitor this new PEG-LEH formulation after i.v. administration in rabbits, we used a previously developed method of
labeling LEH with technetium-99m (99mTc) in a stable fashion combined with scintigraphic imaging to allow for noninvasive determination of changes in organ biodistribution over
the course of the study. This report summarizes our findings
that the addition of PEG-DSPE to the LEH formulation can
increase circulation persistence.
1999
PEG-Liposome-Encapsulated Hemoglobin
667
noted for 99mTc-PEG-LEH appears to be very similar to the
distribution of 99mTc activity noted in the 99mTc-labeled red
blood cell image demonstrated in Fig. 1B. The 24-h 99mTcPEG-LEH image (Fig. 1C) continues to reveal a large amount
of heart activity representing 99mTc-PEG-LEH still in circulation. The increased 99mTc activity visualized in the liver
compared with the 2-h image indicates that the liver is the
major site of PEG-LEH removal. Although continued 99mTc
activity is visualized in the heart at 48 h, the trend of increasing liver 99mTc activity is apparent (Fig. 1D). It is also
apparent that the 48-h image has a lower resolution because
of the decreased 99mTc activity secondary to decay of the
99m
Tc radionuclide (half-life 5 6.1 h).
Figure 2 illustrates the organ distribution of 99mTc-PEGLEH as a percentage of injected dose (% ID) at various times
postinfusion as estimated by image analysis. Figure 2A
shows the distribution of the 99mTc activity in the various
organs uncorrected for the 99mTc activity contributed by the
blood associated with that organ. Figure 2B shows the blood
background-corrected 99mTc activity corresponding to the estimated organ uptake of 99mTc-PEG-LEH over time. Most of
the blood pool-corrected organ uptake of 99mTc-PEG-LEH is
in the liver: 5% ID at the time of completion of the infusion.
Liver uptake decreases to 2% ID by 2 h. This slight decrease
in liver 99mTc activity is apparent by visual inspection of the
images because the heart has more intensity in relation to
the liver at 2 h than it does at 45 min. At 24 and 48 h, the
uptake in the liver is 10% ID. The spleen demonstrated only
measurable uptake after 24 h, and at 48 h, it had increased
99m
Tc activity to only 1.4% ID. The uptake of 99mTc-PEGLEH in other organs was minimal, and image analysis was
not possible.
A clearance profile (Fig. 3) for 99mTc-PEG-LEH was generated by monitoring 99mTc activity in blood samples and
fitting the data to a two-compartment pharmacokinetic
model. The distribution phase rate constant was 0.0005 h21,
and the elimination phase half-life was 65.2 h. The volume of
distribution was 173.9 6 8.4 ml.
Table 2 shows the 48-h organ biodistribution for 99mTc-
Fig. 2. The percent distribution of 99mTc-PEG-LEH at various times over
a 48-h period as determined by region of interest analysis of gamma
camera images. A, Biodistribution of 99mTc-PEG-LEH uncorrected for the
background 99mTc activity contributed by the 99mTc-PEG-LEH circulating
in the blood. B, Blood background-corrected biodistribution of 99mTcPEG-LEH.
PEG-LEH both on a % ID/organ and % ID/g tissue basis
obtained by tissue sampling at necropsy. The blood has 4
times more 99mTc-PEG-LEH (51.3 6 3.4% ID) than the liver
(12.7 6 0.7% ID). Approximately 5% ID is found in the bone
marrow, small bowel, and colon. Only minimal amounts are
noted in the other organs listed. The spleen has the largest
dose based on % ID/g tissue, followed by the blood and the
liver.
Discussion
The PEG-LEH formulation in this study demonstrated
prolonged circulation persistence and offers the potential of
providing extended oxygen delivery. The slow rate of clearance from the blood pool also results in a decreased stress on
the RES. The circulation half-life of 65 h for this PEG-LEH
formulation is greater than 3 times longer than that previously reported for earlier non-PEG-containing LEH formulations (Farmer and Gaber, 1987; Rudolph et al., 1991; Zheng
et al., 1994). In a previous study, after administering the
same volume of a non-PEG-containing LEH formulation to
rabbits in an identical fashion (Rudolph et al., 1991), we
measured a circulation half-life of 18 h. The organ distribution pattern of this non-PEG-containing LEH formulation
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Fig. 1. Gamma camera images of rabbits acquired after a 25% topload
infusion of 99mTc-labeled PEG-LEH. The images were taken at 2 h (A),
24 h (C), and 48 h (D) after infusion of 99mTc-PEG-LEH. B, For comparative purposes, an image of 99mTc-labeled red blood cells (RBCs) acquired
after equilibration at 20 min is shown. The 2-h image of a rabbit receiving
99m
Tc-PEG-LEH is very similar to the 99mTc-RBC image.
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Phillips et al.
Vol. 288
TABLE 2
Tissue biodistribution of 99mTc-PEG-LEH obtained from counting tissue
samples in a scintillation well counter at 48 h
Organ
% ID/Organ
% ID/g Tissue
Blood
Liver
Spleen
Femur
Kidney
Lung
Heart
Brain
Skin
Muscle
Urine
Bowel
Colon
51.27 6 3.44
12.69 6 0.70
1.42 6 0.20
6.18 6 0.14
1.09 6 0.21
0.85 6 0.10
0.11 6 0.01
0.05 6 0.00
1.61 6 0.11
2.30 6 0.28
1.09 6 0.26
5.25 6 0.39
5.34 6 0.89
0.315 6 0.022
0.125 6 0.016
1.179 6 0.064
0.043 6 0.002
0.093 6 0.012
0.092 6 0.012
0.035 6 0.014
0.004 6 0.000
0.006 6 0.001
0.002 6 0.000
0.027 6 0.006
0.013 6 0.002
0.013 6 0.002
Values represent mean 6 S.E.M.
was also very different with rapid liver uptake and apparent
RES saturation. The liver uptake of PEG-LEH was 2% ID at
2 h compared with liver uptake of 16% ID at 2 h with the
non-PEG-containing LEH formulation. In addition, PEGLEH in the present study had a greatly diminished accumulation in the spleen compared with non-PEG-containing
LEH. With PEG-LEH, only 1.4% ID accumulated in the
spleen at 48 h, much less than the 18.1% ID distributed to the
spleen at 24 h with the non-PEG-containing LEH formulation (Rudolph et al., 1991).
Only two previous studies have attempted to prolong the
circulation of LEH by modification of the liposomal surface.
In one study, LEH was surface modified with ganglioside
GM1. This ganglioside was the first molecule described to
increase the circulation persistence of a liposome formulation
(Allen et al., 1989). In the case of LEH, however, ganglioside
GM1 surface modification did not result in an increase in the
circulation persistence of LEH (Goins et al., 1994).
In a second study, LEH was surface modified with 5 mol%
PEG-PE with a PEG-headgroup of molecular weight of 1900
(Zheng et al., 1994). The PEG-LEH formulation containing
phosphatidylcholine/cholesterol/PEG-PE/a-tocopherol
(1:1:0.1:0.02) was compared with non-PEG-containing LEH.
A 50% isovolemic exchange was performed in rats to directly
compare non-PEG-containing LEH with PEG-LEH. The cir-
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Fig. 3. Circulation persistence of 99mTc-PEG-LEH as determined by
scintillation counting of blood samples. Samples were collected over a
48-h period. Top right inset, expanded view of the circulation persistence
over the first 2 h.
culation time for this 5 mol% PEG-LEH formulation was not
extended over non-PEG-containing LEH; both LEH preparations had a circulation half-life of about 15 to 20 h (Zheng et
al., 1994). The lack of an effect on circulation persistence with
5 mol% PEG-LEH formulation compared with the present
study may have been due to the lower mol% of PEG-PE (5
mol% versus 10 mol%). This lack of effect could also be due to
the choice of PEG-headgroup with a molecular weight of 1900
rather than 5000. The formulation of PEG-LEH in the current study contains an approximately 5-fold increase in the
absolute amount of PEG compared with the PEG-LEH formulation from the previous study containing 5 mol% PEG-PE
with a PEG headgroup of 1900 molecular weight. Theoretical
studies have shown that a near-maximum repulsive barrier
(i.e., steric stabilization) is present when a liposome is modified with a 10 mol% PEG phospholipid containing a PEG
headgroup of 5000 molecular weight (Needham et al., 1992).
Most LEH preparations investigated to date have contained 10 mol% of the negatively charged phospholipid,
dimyristoyl phosphatidylglycerol (DMPG), which was used to
improve liposome dispersion and prevent their aggregation
during manufacture (Farmer and Gaber, 1987). The inclusion of PEG-PE in the LEH formulation obviates the need for
DMPG because PEG also significantly reduces liposome aggregation (Yoshioka, 1991; Takahashi, 1995; Sakai et al.,
1997). The removal of DMPG has the added advantage of
further increasing LEH circulation persistence because the
negative charge of DMPG has been associated with an increased rate of liposome opsonization and removal from circulation by the RES (Reinish et al., 1988).
Although this increased PEG content of the present PEGLEH formulation is likely responsible for its increased circulation persistence, the question of increased toxicity from
PEG will need to be addressed. Previous studies indicate that
PEG surface-modified liposomes are still eventually cleared
primarily by the RES located in the liver, spleen, and bone
marrow, although other organs, such as the gut and skin,
have increased uptake (Woodle et al., 1995). Two recent
studies address the effect of large amounts of PEG on the
RES (Eldridge et al., 1996; Conover et al., 1997). In these
studies, morphologic and functional studies did not reveal
any toxic effects of the blood substitute, PEG-conjugated
unencapsulated hemoglobin, on the liver or spleen. Further
investigation of PEG-LEH will be needed to determine
whether large amounts of this formulation produce any significant toxicities.
The use of 99mTc as a label allowed serial dynamic scintigraphic imaging and determination of changes in biodistribution at multiple time points without sacrifice of the animal
until the end of the study. This 99mTc liposome label has been
shown to be extremely stable, with in vitro studies showing
,2% dissociation of 99mTc from the liposome label during
incubation with plasma at 37°C for 90 h (Phillips et al., 1992).
The 99mTc label also allowed determination of actual tissue
biodistribution after sacrifice by counting tissue samples in a
scintillation well counter. High quality statistics are possible
out to 90 h due to the high sensitivity of the scintillation well
counter for counting tissue samples. Generally, the tissue
samples cannot be counted until at least 60 h after the start
of a study so that sufficient time is allowed for the 99mTc to
decay (.1 mCi of activity saturates the electronics of the well
counter and produces excessive dead time).
1999
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Future studies will need to address the oxygen-carrying
capacity of PEG-LEH. For adequate oxygen transport, it
obviously will be important that a blood substitute have an
adequate hemoglobin concentration. The hemoglobin content
of the current preparation is low (1.2 g/dl), and methods to
increase its hemoglobin content need to be studied. Other
methods of manufacturing liposomes may result in an LEH
formulation with increased hemoglobin content. One such
method, the dehydration-rehydration method, has been reported to have significantly increased hemoglobin content
(Brandl and Gregoriadis, 1994). Researchers using this
method have described hemoglobin contents of .10 g/dl.
It will also be important for the hemoglobin in LEH to
remain in a functional state after infusion. Several animal
studies have demonstrated that a substantial portion of both
cross-linked hemoglobin and LEH are converted to met-hemoglobin after infusion (Boer et al., 1993; Phillips et al.,
1997). Using the radionuclide, oxygen-15, we recently demonstrated that after initial administration, LEH carries the
amount of oxygen that would be expected for its hemoglobin
content. However, liposomes encapsulating only bovine hemoglobin gradually lost their ability to carry oxygen over a
24-h period, presumably due to met-hemoglobin conversion.
This occurred even though approximately 50% of the encapsulated hemoglobin remained in circulation (Phillips et al.,
1997). On the other hand, LEH-encapsulating human hemolysate, which contains naturally occurring antioxidants, retained hemoglobin in the oxyhemoglobin state over a 24-h
period and only lost oxygen-carrying capacity due to its removal from the circulation by the RES (Phillips et al., 1997).
These observations establish the important difference between physical half-life and functional half-life of red cell
substitutes and suggest that methods to protect encapsulated hemoglobin from oxidative reactions may prolong the
functional half-life of LEH. Recently, several approaches,
such as the inclusion of artificial enzymatic reduction systems in LEH to decrease met-hemoglobin formation, have
been reported (Ogata et al., 1996; Takeoka et al., 1996). The
ability to coencapsulate hemoglobin protectant systems with
encapsulated hemoglobin gives liposomes a significant advantage over other methods of prolonging hemoglobin circulation such as crosslinking hemoglobin or conjugating hemoglobin with PEG.
Another important approach for decreasing met-hemoglobin formation inside of liposomes may be to genetically engineer hemoglobin that is less susceptible to met-hemoglobin
conversion (Vandegriff, 1995; Olson, 1996). Future research
investigating various approaches of decreasing the rate of
met-hemoglobin formation will be important, especially now
that methods of significantly prolonging the circulation persistence of red cell substitutes are becoming available. The
surface modification of LEH with PEG represents an initial
step in the development of an encapsulated hemoglobinbased red cell substitute with prolonged oxygen-delivery capability.
PEG-Liposome-Encapsulated Hemoglobin
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Send reprint requests to: Dr. William Phillips, Department of Radiology,
University of Texas Health Science Center at San Antonio, 7703 Floyd Curl
Dr., San Antonio, TX 78284. E-mail: [email protected]
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