ARTIFICIAL CELLS, BLOOD SUBSTITUTES, AND BIOTECHNOLOGY Vol. 32, No. 2, pp.

173187, 2004

Pharmacodynamic Study of Polyethylene Glycol Conjugated Bovine Hemoglobin (PEG-bHb) in Rats

Zhigang Bi,1 Xueying He,1 Xiaowei Zhang,1 Yong Jiang,2 Kesen Zhao,2 and Qian Liu1,3,*
Beijing Kaizheng Biotech Developing Ltd., Beijing, P.R. China 2 First Military Medical University, GuangZhou, P.R. China 3 Chinese Academy of Medical Sciences and Peking Union Medical College, Dongcheng District, Beijing, P.R. China
1

ABSTRACT
The purpose of this study was to determine the pharmacodynamics of polyethylene glycol (PEG) conjugation on stroma-free bovine hemoglobin, hemorrhage shock, and exchange transfusion rat models were used. In both rat models, blood pressure increased in 6% PEG-bHb treated animals was significantly higher than dextran 70, even than whole blood (isovolume of hemorrhage) transfusion. Six percent PEG-BHb could ameliorate the micro-circulation of the

*Correspondence: Qian Liu, Beijing Kaizheng Biotech Developing Ltd., 9th10th Floor Biosience Building, 27 Taiping Road, Beijing 100850, P.R. China; E-mail: qianliu@ms.imicams.ac.cn. 173
DOI: 10.1081/BIO-120037826 Copyright & 2004 by Marcel Dekker, Inc. 1073-1199 (Print); 1532-4184 (Online) www.dekker.com

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Bi et al. experimental animals markedly, including reducing the blood viscosity and improving the blood flow. This effect of PEG-bHb was superior to autogenous blood which only recovered 50% blood flow, and theres no effect on blood flow when used isovolumic dextran 70. Tissue oxygenations of rats were evaluated by the oxygen dependent quenching of phosphorescence using an Oxyspot phosphorimeter, and the results showed that capability of oxygen-delivery of PEG-bHb was close to autogenous blood and superior to dextran 70. Based on these effects, the survival rates of animals treated with PEG-bHb were close to that of the whole blood transfusion. And data suggested that half of the hemorrhage transfusion is the reasonable therapeutic dosage. In conclusion, PEG-bHb is an effective blood substitute with powerful tissue oxygenation and blood volume expansion. Key Words: Polyethylene glycol conjugated bovine hemoglobin; Hemorrhage shock; Exchange transfusion; Pharmacodynamics.

INTRODUCTION With the realization that plasma expansion alone was not adequate replacement for blood in the treatment of hemorrhage, the limitations of liquid RBCs such as the source and storage, requirement for crossmatching and transmission of diseases, seeking effective blood substitutes has become an urgent task since the World War II (Ketcham and Cairns, 1998). Bovine erythrocytes are one source of hemoglobin that has shown potential in the development of hemoglobin based oxygen carriers (HBOC). Bovine Hb is cheaper than human Hb, doesnt require 2,3-diphosphoglycerate to lower its oxygen affinity, and can enhance delivery of oxygen at a lower pH because of Bohr effect (Benesch et al., 1972). However, unmodified stroma-free hemoglobin are rapidly cleared from the body and can cause renal toxicity (Center for Biologics Evaluation and Research, 1991). Structural modification of hemoglobin can attenuate some of these unfavorable attributes while preserving its oxygen carrying ability (Squires, 2002). Proteins pegylation, which is conjugated polyethylene glycol (PEG) to proteins, has been demonstrated to reduce renal filtration dramatically, prolong blood circulation, and reduce any potential immune or allergic reactions (Veronese, 2001). PEG-bHb is developed for intravenous use as an oxygen-carrying solution by Kaizheng Biotech, and its pharmacodynamics will be

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evaluated by shock resuscitation and exchange transfusion rat models in this contribution.

MATERIALS AND METHODS Animals Sprague-Dawley rats (mixed gender, 180220 g), provided by Experimental Animals Center of the First Military Medical University of China (Certification No.: 2000A059).

Test Solutions PEG-bHb (6% bovine hemoglobin wt/v solution, methemoglobin <5%, endotoxin <1.0 EU/mL, osmolality 330 30 mOsm, pH 7.4 0.2) was formulated in buffer consisting of 150 mmol/L NaCl, 5 mmol/L NaHCO3, 4 mmol/L Na2HPO4, and 1 mmol/L NaH2PO4 and stored at 35 C. The dextran 70 was purchased from Sigma.

Animal Models In both models, rats were anesthetized using 13.3% urethane and 0.5% chloralose (0.6 mL/kg, i.p.), and placed on a heating pad with their body temperature maintained at approximately 37 C. In hemorrhagic shock model, MAP of rats was maintained at 40 mmHg for 1 h by blood controlled withdrawal, then transfused with PEG-bHb and other fluids. In the exchange model, rats received a 50% exchange transfusion of their estimated blood volume (weight of animal (kg) 60 mL/kg 50%). Rats of two models were randomized into Seven treatment groups respectively (Table 1).

Pharmacodynamics Measurement Mean arterial blood pressure (MAP) and heart rate (HR) were monitored every 10 min, beginning 2 h before surgery and for 3 h after transfusion. Blood samples were withdrawn to test Cl, pH, PCO2, PO2, and viscosity. Micro-circulation of rat spinotrapezius muscle was

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Groups shock model S1 S2 S3 S4 S5 S6 S7 Groups exchange model E1 E2 E3 E4 E5 E6 E7

Bi et al. The treatment groups of rats.

No. in groups 26 34 8 36 42 39 35

No. in groups 26 34 7 38 59 43 35

Resuscitation fluids No resuscitation fluids were given Whole blood (isovolume of hemorrhage) Whole blood (1/2 volume of hemorrhage) PEG-bHb (1/2 volume of hemorrhage) PEG-bHb (isovolume of hemorrhage) PEG-bHb (1.5-fold volume of hemorrhage) Dextran (isovolume of hemorrhage)

observed (Gray, 1973), including the velocity and volumetric flow rate and the diameter of the microvessels. Surface tissue oxygen tension was measured by the oxygen dependent queching of phosphorescence using an OxyspotTM phosphorimeter (Medical Systems Corp, Greenvale, NY). A dosage of 2 mg/100 g b.wt of phosphorescent probe was administered via vein (Conover et al., 1999). Data were recorded and analyzed 60 min before and after shock and 0, 30, 60, 120, 180 min after resustation. Statistical Analysis The differences among treatment groups were assessed by one-way ANOVA. Multiple comparisons, when significant differences existed, were determined by least significant differences techniques. Statistical significance was defined as p < 0.05 to reject a null hypothesis. Statistical analysis was conducted with SPSS 10.0 software program. RESULTS Survival PEG-bHb and whole blood-treated groups showed significantly higher survival rate ( p < 0.05) than the no fluid group and dextran group at 24 h in the exchange model. Resuscitation effects were best by half volume hemorrhage PEG-bHb infusion in shock (S4) and exchange (E4) models in rats and infusion isovolume and 1.5-fold volume hemorrhage PEG-bHb showed lower survival rate (Figs. 1 and 2).

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90 80

177
S1 S2 S3

Survival(%)

70 60 50 40 30 20 10 0 24 h 48 h 72 h

S4 S5 S6 S7

Time

Figure 1. Survival of rats at 24, 48, and 72 h in shock model. (View this art in color at www.dekker.com.)

100

Survival(%)

80 60

40 20 0
24h 48h 72h

E1 E2 E3 E4 E5 E6 E7

Time

Figure 2. Survival of rats at 24, 48, and 72 h in exchanged transfusion model. (View this art in color at www.dekker.com.)

Cardiovascular Monitoring Basal conscious MAP and HR were similar among all groups in both models examined and were consistent with the normal values of the rat. MAP decreased with volume controlled hemorrhage in model rats, then

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140 120

Bi et al.
S1 S2 S3 S4 S5 S6 S7

MAP(mmHg)

100 80 60 40 20 0 Normol

Shock Transfusion 30 min 0 min

60 min

120 min

180 min

Time

Figure 3. The MAP of rats in hemorrhage shock model. Blood pressure increased when treatment fluids infused. (View this art in color at www. dekker.com.)

increased until infusion of treatment fluids. PEG-bHb could maintain MAP just as autogenous blood throughout the study period and dextran had lower ability to raise MAP after rat shock. HR showed a positive correlation with MAP (showed in Figs. 36).

Blood Measurement Considering the fact that the concentration of Cl modulates the PEG-bHb capability to release oxygen to the tissues, we detected chloride ion in rat blood. The result did not show any significant [Cl] difference in rat among groups after fluids infusion and the normal states in two models (Tables 2 and 3). Arterial pH values decreased progressively during hemorrhage in all treatment groups (S17, E17), and rectified to normal level after transfusion treatment fluids at 30 min, except for the groups of no infusion. The PCO2 and PO2 values varied slightly during the study. Whole blood viscosity of all groups were evaluated during experiments at different shearing stress of 5 and 200 p/s. Infusion of PEG-bHb could lower the viscosity of experimental rats obviously. Blood viscosity came back to normal through autogenous blood transfusion and decreased mildly by dextran infusion (Tables 4 and 5).

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179
S1 S2 S3 S4 S5 S6 S7

HR (beats/min)

400

350

300

250

200 Normal Shock 60min 0 min

30 min

60 min

120 min

180 min

Time
Figure 4. The heart rate of rats in hemorrhage shock model. The heart rate increased with MAP elevation. (View this art in color at www.dekker.com.)

140 120 100 E1 E2 E3 E4 E5 E6 E7

MAP (mmHg)

80 60 40 20 0 Normal

exchange 0 min

30 min

60 min

120 min

180 min

Time

Figure 5. MAP of rats in exchange transfusion model. Efficacy in blood pressure recovery of PEG-bHb was outstanding. (View this art in color at www.dekker.com.)

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500 450

Bi et al.
E1 E2 E3 E4 E5 E6 E7

HR(beats/min)

400 350 300 250 200 Normal

Exchange 0 min

30 min

60 min

120min

180min

Time

Figure 6. Heart rate of rats in exchange transfusion model. (View this art in color at www.dekker.com.)

Table 2.

Concentration change of Cl of rats in shock model (mmol/L, x s ). After infusion

Groups (no.) S1 S2 S4 S5 S6 S7 (16) (15) (8) (30) (26) (21)

Normal state 110.69 4.84 119.96 7.32 114.25 8.32 116.76 13.38 116.95 13.39 120.93 13.59

30 min 114.86 5.13 123.51 7.76 122.05 12.52 121.76 15.97 122.56 13.51 132.02 14.47

180 min 111.88 5.41 118.38 7.07 121.05 7.85 120.87 14.48 117.27 7.31 125.61 14.44

There is no significant difference among groups.

Microvascular Variables Microvascular Diameter Sixty minutes after shock, a variable narrowing was seen in the arteriolar vessels, which was recovered by transfusion, and the venular vessles diameter didnt show any change. Theres no considerable shrink in arteriolar and venular vessles throughout exchange transfusion in rats.

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Table 3. Concentration change of Cl of rats in exchange transfusion model (mmol/L, x s ). After exchange transfusion Groups (no.) E1 E2 E4 E5 E6 E7 (15) (21) (7) (46) (32) (22) Normal state 118.44 9.87 118.91 9.63 121.69 10.57 118.74 7.63 117.36 10.56 123.48 7.85 30 min 124.64 15.53 127.09 8.62 123.13 6.43 123.53 10.72 124.82 8.32 133.37 15.47 180 min 117.45 0.21 118.07 5.78 121.79 8.65 120.36 10.86 125.80 12.33 128.85 12.04

Table 4. Changes of blood viscosity of shock model rats at different shearing stress of 5 p/s and 200 p/s (x s).
5 p/s Groups (no.) S1 S2 S4 S5 S6 S7 (26) (34) (36) (42) (39) (35) After infusion Normal 6.78 1.31 9.13 2.28 7.12 1.60 9.43 1.14 7.63 1.74 8.94 2.97 30 min 6.33 1.05 9.14 2.17 4.55 0.86 4.02 1.19 3.51 0.69 3.75 1.10 180 min 5.55 1.69 8.24 3.41 3.51 0.46 3.41 0.48 3.65 0.44 3.70 0.82 Normal 2.91 0.41 3.15 0.31 2.69 0.23 2.66 0.55 2.88 0.50 3.23 1.21 200 p/s After infusion 30 min 1.91 0.33 3.32 0.60 1.96 0.11 1.70 0.35 1.63 0.37 1.94 0.34 180 min 2.22 0.31 2.97 0.45 1.76 0.07 1.77 0.06 1.77 0.13 1.87 0.21

Table 5. Changes of blood viscosity of exchange model rats at different shearing stress of 5 p/s and 200 p/s (x s).
5 p/s Groups (no.) E1 E2 E4 E5 E6 E7 (26) (34) (38) (59) (43) (35) After infusion Normal 6.44 2.55 9.33 2.76 9.51 3.11 8.98 1.81 8.69 3.03 8.59 1.88 30 min 6.02 2.6 8.39 1.84 4.26 0.74 4.56 0.37 4.33 0.21 5.46 0.84 180 min 6.89 2.35 7.49 1.91 3.86 0.69 4.05 1.54 4.37 0.41 5.81 1.66 Normal 2.59 0.7 3.29 0.68 3.06 0.59 3.32 0.55 3.19 0.91 3.19 0.45 200 p/s After infusion 30 min 2.60 0.94 2.95 0.36 2.01 0.19 2.17 0.13 1.95 0.16 2.34 0.26 180 min 2.97 0.80 2.82 0.42 1.93 0.31 2.08 0.17 1.93 0.2 2.45 0.57

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S1 S2 S4 S5 S6 S7

Flow rate(mm/s)

8 6 4 2 0 Normal Shock 0 min 60 min Infusion 0 min 30 min

60 min

120 min

Time
Figure 7. The flow rate of arteriole in hemorrhage shock model rats. (View this art in color at www.dekker.com.)

Blood Flow Volumetric flow was dramatically reduced in arterioles and venules in the two models. The flow rate recovered nearly to normal by infusion of PEG-bHb, while increased to half of normal with autogenous blood transfusion. Along with the increase of flow rates, blood flow increased compared to hemorrhage. Dextran couldnt ameliorate microvascular flow rate of the model rats (Figs. 710).

Tissue Oxygenation To evaluate the oxygen delivery capability of PEG-bHb directly, oxygen tension of liver, muscle, and small intestine was measured by the oxygen dependent quenching of phosphorescence. The results showed that tissue oxygen tension decreased significantly after shock, and could be increased by reinfusion of whole blood and PEG-bHb, while dextran had less capability to raise tissue oxygen tension ( p < 0.05, compared to PEG-bHb and whole blood) (Tables 68). In exchange transfusion model, PEG-bHb had the middle oxygen delivery ability compared with whole blood and dextran (Table 9).

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183
S1 S2 S4 S5 S6 S7

Flow rate(mm/s)

2.5 2 1.5 1 0.5 0 Normal Shock 0 min 60 min

Infusion 0 min

30 min

60 min

120 min

Time

Figure 8. The flow rate of venula in hemorrhage shock model rats. The blood flow rate of venula recovered nearly to normal level when PEG-bHb transfused. (View this art in color at www.dekker.com.)

10 9 8

E1 E2 E4 E5 E6 E7

Flow rate(mm/s)

7 6 5 4 3 2 1 0 Normal Exchange 0 min 30 min 60 min 120 min

Time
Figure 9. The flow rate of arteriole in exchange transfusion model rats. The blood flow rate of arteriole recovered nearly to normal level when PEG-bHb transfused. (View this art in color at www.dekker.com.)

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4 3.5 3

Bi et al.
E1 E2 E4 E5 E6 E7

Flow rate(mm/s)

2.5 2 1.5 1 0.5 0 Normal Exchange 0 min 30 min 60 min 120 min

Time
Figure 10. The flow rate of venula in exchange transfusion model rats. (View this art in color at www.dekker.com.)

Table 6. Oxygenation of liver throughout the study in shock model rats (% change from basal, x s).
Shock Groups S1 S2 S4 S5 S6 S7 0 min 92.56 3.13 94.49 3.77 93.89 4.23 92.72 7.24 95.33 1.18 95.08 3.66 60 min 95.50 0.34 98.07 0.33 97.19 2.37 93.03 5.41 97.66 1.13 95.71 3.37 0 min 96.95 2.66 7.57 19.47 49.24 22.04 43.48 24.74 40.16 12.47 59.11 21.17 After reinfusion 30 min 94.55 0.51 26.30 15.39 58.12 7.51 43.35 23.12 38.16 5.10 73.47 17.66 60 min 95.46 0.46 24.98 17.91 59.15 6.58 55.37 15.74 40.15 18.53 78.75 20.63

DISCUSSION To evaluate the pharmacodynamics of PEG-bHb, hemorrhage shock and 50% exchange transfusion models of rats were used in our study. Due to the characteristic of PEG-bHb (e.g., the viscosity and colloid osmolality), its efficiency depends on the dosage used. Three different dose groups were then designed. In two models, the survival rates were higher in 1/2 hemorrhage volume transfusion group (S4, E4) than in

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Table 7. Oxygenation of muscle throughout the study in shock model rats (% change from basal, x s).
Shock Groups S1 S2 S4 S5 S6 S7 0 min 62.06 5.42 69.72 14.67 67.96 13.46 77.33 10.02 75.76 14.13 75.50 18.45 60 min 69.02 11.30 81.41 6.40 74.35 28.22 89.78 8.60 84.99 6.11 86.37 12.79 0 min 69.02 11.30 17.54 5.46 35.75 29.04 22.09 27.36 15.93 7.19 73.51 28.33 After reinfusion 30 min 85.81 5.84 7.97 13.44 18.62 20.94 16.58 14.77 13.71 12.45 60.36 20.99 60 min 82.06 6.97 26.65 9.28 30.85 17.48 22.08 12.88 18.32 31.67 58.27 18.01

Table 8. Oxygenation of intestine throughout the study in shock model rats (% change from basal, x s).
Shock Groups S1 S2 S4 S5 S6 S7 0 min 79.55 16.85 81.83 10.47 78.17 20.75 76.28 17.02 79.00 18.70 90.04 5.69 60 min 77.18 8.69 85.14 8.21 86.54 13.77 80.60 14.92 81.37 19.46 91.08 8.00 0 min 77.18 8.69 36.36 30.75 45.24 32.12 24.63 36.88 41.18 19.77 63.63 28.30 After reinfusion 30 min 85.23 7.41 46.86 22.48 48.87 34.87 48.76 21.36 44.10 22.35 70.56 28.44 60 min 84.49 12.27 39.77 37.76 60.59 25.55 53.43 20.42 46.52 31.46 72.85 28.57

groups where isovolume and 1.5-fold volume of PEG-bHb were used. Compared with whole blood and dextran transfusion controls, the results of rats survival showed that the PEG-bHb (when half of hemorrhage volume were used) had the approximate resuscitation effect as the whole blood, and was superior to dextran significantly. MAP and HR are the indexes to demonstrate the function of cardiovascular system. MAP of rats in two models came back to the normal level nearly when PEG-bHb transfused and even superior to the autogenous blood, which suggests that PEG-bHb has excellent volume expansion function. HR lowered in rats when MAP decreased, maybe lack of functional reflex regulation in the anesthetized rats, and HR recovered with MAP increased to the normal level. Blood viscosity is an important factor determing microvascular flow, and RBC is the key element to maintain the blood viscosity. Cell-free

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Table 9. Oxygenation of tissues throughout the study in exchange model rats (% change from basal, x s).
After transfusion Tissues Liver Groups S1 S2 S4 S5 S6 S7 Muscle S1 S2 S4 S5 S6 S7 Intestine S1 S2 S4 S5 S6 S7 0 min 98.02 1.12 30 min 94.49 6.60 1h 1.5 h 2h 97.77 2.67 62.45 9.80 70.87 4.09 73.50 4.60 91.30 4.00 96.01 6.50

84.47 24.61 97.26 3.36 52.92 10.87 60.76 8.58 67.58 4.60 71.86 6.58 91.37 4.55

13.24 14.90 32.23 13.06 43.34 10.80 52.23 13.56 57.67 10.72 31.20 11.05 44.49 9.87 46.47 8.30 80.01 3.84 38.76 14.84 49.70 11.20 64.08 8.95 58.90 10.73 67.01 8.06 79.54 7.26 81.79 10.3

80.31 20.27 77.58 21.78 88.62 12.94 95.08 6.77

11.36 15.96 12.77 29.19 29.92 15.73 41.89 10.68 52.47 12.84 20.32 24.89 33.24 24.56 41.51 24.74 49.66 15.24 61.14 11.98 19.46 28.37 31.66 16.43 44.01 9.13 54.98 11.11 63.50 12.84 16.27 21.57 29.03 19.96 35.15 15.72 51.97 20.16 67.41 16.37 65.98 20.63 59.74 11.85 70.04 13.38 78.62 11.36 81.34 11.94 75.11 29.86 82.02 29.50 91.95 7.57 93.72 7.38 95.20 7.34 52.02 10.37 23.89 22.45 26.90 36.16 32.06 19.25 37.07 24.75 40.18 25.53 39.98 14.54 40.39 13.01 46.46 12.59 55.08 8.42 47.06 4.89 45.65 12.21 46.74 21.97 50.14 15.44 50.56 15.30

17.45 14.49 31.73 25.41 39.88 19.30 43.02 17.02 46.05 7.99 63.05 15.69 72.33 13.03 72.62 20.74 70.66 33.37 70.46 31.58

PEG-bHb has lower viscosity and dilution effect when transfused due to higher colloid osmolality, so PEG-bHb would induce blood viscosity decrease with increased infusion dose. The lower viscosity of blood will reduce the flow resistance to increase tissue blood flow, but theres information argued that moderate viscosity is necessary to maintain blood shearing stress to the vascular wall, which is related with NO synthesis and release, and NO could prevent microvascular contract (de Figueiredo, 1998). This may be one of the reasons why the resuscitation effect was better when 1/2 hemorrhage volume transfused than isovolume and 1.5-fold volume used. Microvascular observation approved that appropriate PEG-bHb infusion could ameliorate the microvascular flow. Tissue oxygenation measurement provided the direct proof of the oxygen delivery capability of PEG-bHb. The oxygen tension of liver, muscle, and intestine decreased dramatically when rats in shock or 50% exchange transfusion, and raised with PEG-bHb transfusion. Although the recovery effect was limited, it was obviously comparative to whole

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blood and superior to dextran. In addition, theres another evidence that the survival time of rabbits of 95% exchange transfusion by PEG-bHb was above 25 h, which confirms the oxygen delivery capability of PEGbHb (data not published in our corporation). In conclusion, a 6% solution of PEG-bHb can adequately deliver oxygen to tissue and has comparative resuscitation effect with the whole blood in both hemorrhage shock and 50% exchange transfusion rat models. PEG-bHb is an effective blood substitutes.

ACKNOWLEDGMENTS Contract grant sponsor: National High-tech program.

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