Polyethylene glycolized bovine hemoglobin oxygen carrier and preparation method and application thereof

Abstract

The application provides a polyethylene glycolized bovine hemoglobin oxygen carrier and a preparation method and application thereof, relates to the technical field of blood substitutes, and the bovine hemoglobin is modified with methoxypolyethylene glycol vinyl sulfone on the surface. 50 The polyethylene glycolized bovine hemoglobin oxygen carrier has a high P (50% oxygen saturation oxygen partial pressure), can effectively carry and release oxygen, and has a wide application prospect as a blood substitute.

Description

Technical Field

[0001] This invention relates to the field of blood substitute technology, and in particular to a polyethylene glycol-modified bovine hemoglobin oxygen carrier, its preparation method, and its application. Background Technology

[0002] In recent years, with the rapid growth in clinical blood demand, the contradiction between blood supply and demand has become increasingly prominent. Traditional blood transfusion plays an important role in medical care, but it also has various drawbacks, mainly reflected in: (1) due to the existence of the window period, there is a risk of pathogen transmission during blood transfusion, such as the presence of pathogens like HIV and hepatitis B virus in the blood; (2) blood shortage, insufficient reserves in clinical blood banks, especially for patients with rare blood types who may lose their lives due to lack of compatible blood; (3) natural red blood cells have a short storage time, only 5-6 weeks in blood banks at 4°C, and the transportation conditions are harsh; (4) crossmatching is required before transfusion, which is not conducive to emergency rescue. This makes it impossible for the already scarce blood supply to meet the needs of clinical treatment and emergency rescue, leading to a major challenge for traditional blood transfusion therapy and limiting the application of natural blood in emergency treatment of massive blood loss.

[0003] Blood substitutes with oxygen-carrying / emission functions have emerged and become a research hotspot both domestically and internationally. Blood substitutes partially replace blood by mimicking the structure and function of blood components; they can be stored for long periods, avoiding blood-borne diseases; and they offer advantages such as abundant supply, convenient storage and transportation, and no need for cross-matching, making them a necessary supplement to clinical blood supplies. Blood substitutes mainly include two categories: fluorocarbons and hemoglobin-based oxygen carriers (HBOCs). Due to the persistent toxicity and metabolic problems of fluorocarbons, HBOCs have become the primary type of blood substitute. Hemoglobin (Hb) is a tetrameric protein composed of four subunits (ααββ) with oxygen-carrying and oxygen-emission functions. HBOCs, based on human or animal-derived Hb, remove its antigenicity and toxicity through chemical modification, thus safely and effectively replacing red blood cells in their oxygen-carrying / emission functions, making them an ideal blood substitute.

[0004] Free hemoglobin easily permeates the vascular endothelium, binds to vasodilators, causing vasoconstriction, vasoactivity, and elevated blood pressure. Hemoglobin tetramers readily depolymerize into dimers, losing their oxygen-carrying / emission function. These dimers, after being filtered by the kidneys, accumulate in the renal tubules, leading to tubular necrosis, making them unsuitable for direct infusion. Red blood cell substitutes with oxygen-carrying / emission functions are primarily produced by chemically modifying, polymerizing, or cross-linking matrix-free hemoglobin, or by encapsulating it in polymer microcapsules. This allows them to mimic the oxygen-carrying and emission capabilities of hemoglobin in natural red blood cells, and they can be safely and effectively used in the human body.

[0005] Polyethylene glycol (PEG) is a neutral, non-toxic polymer with good biocompatibility. The terminal hydroxyl groups of PEG can be derived into various reactive functional groups, allowing for covalent modification of certain amino acid residues in proteins. Since Abuchowski et al. first used PEG to modify bovine serum albumin in 1977, PEG has been widely used in the modification of protein drugs. PEG modification of hemoglobin increases its molecular weight and forms a protective layer around the hemoglobin molecule. Therefore, PEG modification can maintain the structural integrity of the oxygen-binding sites of hemoglobin, reduce phagocytosis by the reticuloendothelial system, desensitize the immunogenicity and antigenicity of hemoglobin, increase its circulating half-life in vivo, and reduce potential allergic and immune reactions. Besides its use in acute blood loss or hemorrhagic shock, potential indications for PEG-modified hemoglobin include surgical blood preparation, anemia and sickle cell anemia, in vitro tissue perfusion, local ischemia, and as an adjuvant in tumor therapy (Biochimica et Biophysica Acta 2008, 1784:1395–1401).

[0006] PEG-modified hemoglobin can effectively eliminate vasoactivity, as well as effectively expand volume and carry oxygen. This suggests that PEG-modified hemoglobin can serve as a potential blood substitute. Related PEG-modified hemoglobin products, such as the PEGylated human hemoglobin (trade name Hemospan, also known as MP4) developed by Sangart in the United States, have entered Phase III clinical trials. The preparation process of MP4 is as follows: firstly, 2-iminothione is used as a cross-linking agent to react with the amino groups on the surface of human hemoglobin to generate thiol groups. Then, the thiol groups react with PEG maleimide with a molecular weight of 5 kDa to obtain MP4. However, the obtained MP4 has the following drawbacks: 1) MP4's P... 50 The value is very low, about 4-6 mmHg, indicating that MP4 cannot effectively release oxygen; 2) The CS bond formed by the reaction of maleimide and thiol is unstable. For example, under alkaline conditions (pH 9.0), PEGylated proteins based on the maleimide-thiol reaction are prone to PEG dissociation (Electrophoresis 2015, 36: 371-374); under physiological pH and normal temperature conditions, thiol-containing reagents can dissociate the PEG of PEGylated proteins based on the maleimide-thiol reaction (Bioconjugate Chemistry 2011, 22(10): 1946-1953).

[0007] Human hemoglobin is derived from expired human blood, and there is currently a shortage of expired blood; furthermore, bovine hemoglobin (bHb) has a structure similar to human hemoglobin, and its P... 50The value is approximately 28 mmHg, which is much higher than the P of human hemoglobin. 50 The oxygenation value is 14 mmHg. Therefore, some hemoglobin oxygen carriers are prepared using bHb. For example, Enzon Pharmaceuticals in the United States developed PEGylated bovine hemoglobin, which is obtained by directly reacting 5 kDa succinimide ester PEG with the amino group of bHb. The main drawback of this product is its excessively high colloid osmotic pressure; the colloid osmotic pressure of 40 mg / mL PEGylated hemoglobin exceeds 200 mmHg. Excessive osmotic pressure can lead to cell and tissue dehydration, which is detrimental to blood transfusion. Therefore, it is necessary to provide a new PEGylated hemoglobin carrier and its preparation method.

[0008] In view of this, the present invention is proposed. Summary of the Invention

[0009] The purpose of this invention is to provide a polyethylene glycol-modified bovine hemoglobin oxygen carrier, its preparation method, and its application, thereby solving the problem of polyethylene glycol-modified hemoglobin P in the prior art. 50 Problems include very low pH values, ineffective oxygen release, or excessively high colloid osmotic pressure. The PEG-bHb of this invention has a high pH value. 50 It can effectively carry and release oxygen.

[0010] The technical solution provided by this invention is as follows:

[0011] In one aspect, the present invention provides a polyethylene glycol-modified bovine hemoglobin oxygen carrier, wherein the surface of the bovine hemoglobin is modified with methoxy-PEG vinyl sulfone. Specifically, methoxy-PEG vinyl sulfone is covalently linked to the surface of the bovine hemoglobin. The present invention uses methoxy-PEG vinyl sulfone as a modifier, which helps to improve the stability of the linkage between PEG and bovine hemoglobin.

[0012] In one embodiment, the bovine hemoglobin has been thiolated prior to the modification with methoxy polyethylene glycol sulfone. In a specific embodiment, the bovine hemoglobin is thiolated by reacting with 2-iminothione.

[0013] In one embodiment, the P of the polyethylene glycol-modified bovine hemoglobin of the present invention 50 The value is not less than 10 mmHg; the colloid osmotic pressure of the polyethylene glycol-modified bovine hemoglobin in a colloidal solution with a protein concentration of 40 mg / mL is not less than 50 mmHg. 50 The value is the partial pressure of oxygen when hemoglobin is 50% saturated or the partial pressure of oxygen when blood oxygen saturation is 50%.

[0014] In one embodiment, the Hill coefficient of the polyethylene glycol-modified bovine hemoglobin oxygen carrier of the present invention is 1.9.

[0015] In another aspect, the present invention provides a method for preparing the polyethylene glycol-modified bovine hemoglobin oxygen carrier, comprising: mixing and reacting bovine hemoglobin with methoxy polyethylene glycol sulfone to obtain polyethylene glycol-modified bovine hemoglobin, preferably, the bovine hemoglobin is subjected to thiolation treatment.

[0016] In one embodiment, the present invention uses bovine hemoglobin as raw material, 2-iminothion as a connecting bridge, and methoxyPEG vinyl sulfone as a modifier to modify the surface of bovine hemoglobin.

[0017] In one embodiment, the thiolation method involves reacting 2-iminothione with bovine hemoglobin to convert the amino groups of the bovine hemoglobin into thiol groups.

[0018] Preferably, the bovine hemoglobin is dissolved in PBS buffer and then mixed with 2-iminothion dissolved in PBS buffer to obtain thiolated bovine hemoglobin.

[0019] Further, the molar ratio of the bovine hemoglobin to the 2-iminothion is 1:(5-40); the preferred molar ratio is 1:(15-25).

[0020] Furthermore, the reaction is carried out overnight at 4°C;

[0021] Furthermore, glycine is added to terminate the mixing reaction.

[0022] In a specific preferred embodiment, the amino group of the bovine hemoglobin is converted to a thiol group by: dissolving bovine hemoglobin in PBS buffer (pH 7.4), and then mixing it with 2-iminothione dissolved in PBS buffer (pH 7.4) at a molar ratio of 1:20. The reaction is carried out overnight at 4°C, and the reaction is terminated by adding excess glycine; unreacted 2-iminothione and glycine are removed by dialyzing to obtain thiolized bovine hemoglobin.

[0023] In one embodiment, the thiolized bovine hemoglobin is mixed with the methoxy polyethylene glycol sulfone in a molar ratio of 1:(5-40), preferably 1:(35-50, to obtain crude polyethylene glycol-modified bovine hemoglobin.

[0024] Preferably, the molecular weight of the methoxy polyethylene glycol ethylene sulfone is 2-40 kDa;

[0025] Further, the molecular weight of the methoxy polyethylene glycol ethylene sulfone is 3-10 kDa; more preferably, the molecular weight of the methoxy polyethylene glycol ethylene sulfone is 5 kDa.

[0026] Furthermore, the reaction is carried out overnight at 4°C.

[0027] In a specific preferred embodiment, the PEGylation of thiolized bovine hemoglobin is performed as follows: bovine hemoglobin is dissolved in PBS buffer (pH 7.4), and then mixed with methoxy-PEG vinyl sulfone dissolved in PBS buffer (pH 7.4) at a molar ratio of 1:40. The mixture is reacted overnight at 4°C to obtain PEGylated bovine hemoglobin oxygen carrier.

[0028] In one embodiment, the method further includes purifying the crude PEGylated bovine hemoglobin; preferably, size exclusion chromatography is used to purify the crude PEGylated bovine hemoglobin.

[0029] In one specific implementation, the purification process involves loading crude PEGylated bovine hemoglobin onto a Superdex 200 gel filter column (1.6cm × 60cm) for elution, using PBS buffer (pH 7.4) as both equilibration and elution buffer, and detecting and collecting the elution peak corresponding to the PEGylated bovine hemoglobin at 280nm to obtain the PEGylated bovine hemoglobin oxygen carrier.

[0030] In another aspect, the present invention provides the use of the polyethylene glycol-modified bovine hemoglobin oxygen carrier in the preparation of blood substitutes and / or oxygen carriers.

[0031] In one embodiment, the polyethylene glycol-modified bovine hemoglobin oxygen carrier partially or completely replaces red blood cells in carrying or releasing oxygen during blood transfusion. The blood transfusion process includes transfusions caused by trauma, blood loss, anemia, surgery, etc.

[0032] Beneficial effects:

[0033] (1) This invention provides a PEGylated bovine hemoglobin oxygen carrier based on mercapto-ethylene sulfone modification. The PEGylated bovine hemoglobin (PEG-bHb) of this invention has high P 50 (Partial pressure of oxygen at 50% oxygen saturation) can effectively carry and release oxygen.

[0034] (2) The present invention uses methoxyPEG vinyl sulfone as a modifier, which helps to improve the stability between the PEG and bovine hemoglobin linkage;

[0035] (3) The PEGylated bovine hemoglobin oxygen carrier obtained by the present invention has a low osmotic pressure, which is beneficial for blood transfusion and has broad prospects as a blood substitute.

[0036] (4) The preparation method of the present invention is simple, efficient, easy to operate and promote. Attached Figure Description

[0037] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0038] Figure 1 The chemical reaction formula for preparing the PEG-bHb of the present invention is shown;

[0039] Figure 2 The results show the analysis of PEG-bHb prepared under different reaction conditions by SDS-PAGE electrophoresis. Lane 1: standard protein; Lane 2: bHb; Lanes 3-11: PEG-bHb with molar ratios of bHb:2-iminothionane:PEG of 1:10:20, 1:20:20, 1:30:20, 1:10:30, 1:20:30, 1:30:30, 1:10:40, 1:20:40, and 1:30:40, respectively.

[0040] Figure 3 The image shows the elution peak spectrum when purifying the PEG-bHb of this invention using a Superdex 200 semi-preparative gel filtration column.

[0041] Figure 4 The image shows the elution peak spectrum of PEG-bHb of this invention analyzed using an analytical Superdex 200 gel filtration column.

[0042] Figure 5 The image shows an electrophoresis diagram of the PEG-bHb of the present invention analyzed by SDS-PAGE. Lane 1: standard protein, Lane 2: bHb, Lane 3: PEG-bHb.

[0043] Figure 6 The particle size distribution diagram of PEG-bHb of the present invention is shown;

[0044] Figure 7 The circular dichroism spectrum of PEG-bHb of the present invention is shown;

[0045] Figure 8 The UV-Vis spectrum of PEG-bHb is shown.

[0046] Figure 9 The diagram shows the oxygen binding and release curves of PEG-bHb of the present invention. Detailed Implementation

[0047] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0048] Example 1: Optimization of the preparation reaction of PEG-bHb

[0049] The yield of PEG-bHb can be improved by optimizing the molar ratio of bHb, 2-IT, and PEG. The changes in PEG-bHb under different molar ratios were detected by SDS-PAGE. Figure 2 As shown, the yield of PEG-bHb gradually increased when the molar ratio of bHb:IT:PEG was 1:10:20 (lane 3), 1:20:20 (lane 4), and 1:30:20 (lane 5). This indicates that increasing the molar ratio of IT:bHb helps to improve the yield of PEG-bHb when the molar ratio of bHb:IT:PEG remains constant. The yield of PEG-bHb also gradually increased when the molar ratio of bHb:IT:PEG was 1:10:30 (lane 6), 1:20:30 (lane 7), and 1:30:30 (lane 8). This indicates that increasing the molar ratio of PEG:bHb helps to improve the yield of PEG-bHb. When the molar ratios of bHb:IT:PEG were 1:10:40 (lane 9), 1:20:40 (lane 10), and 1:30:40 (lane 11), the yield of PEG-bHb gradually increased. However, the yields of PEG-bHb at molar ratios of 1:20:40 and 1:30:40 showed no significant change. Therefore, this invention uses a molar ratio of 1:20:40 for the preparation of PEG-bHb.

[0050] This invention uses a chemical modification method to covalently link polyethylene glycol (PEG) with a molecular weight of 5 kDa to bovine hemoglobin (bHb) to prepare a bovine hemoglobin-based oxygen carrier, namely PEGylated bovine hemoglobin oxygen carrier (PEG-bHb), which can be used as a red blood cell substitute and oxygen carrier.

[0051] The method for preparing this PEG-bHb specifically includes the following steps:

[0052] (1) Thiolization of bHb: 2 mL of bHb (60 mg / mL) solution was dialyzed against 1 L of PBS buffer (pH 7.4), with the buffer being replaced every 4 hours for a total of two replacements. Subsequently, bHb was diluted with PBS buffer (pH 7.4) to adjust its concentration to 64 mg / mL. 2-Iminithiothion dissolved in PBS buffer (pH 7.4) was mixed with bHb at a molar ratio of 20:1, and the mixture was reacted at 4 °C for 8 hours. Figure 1 As shown, 2-iminothionane can react with the amino group of bHb to generate a thiol group.

[0053] (2) Preparation of PEGylated bHb: The thiolized bHb obtained in step (1) was mixed with methoxyPEG vinyl sulfone with a molecular weight of 5 kDa at a molar ratio of 1:40 and reacted overnight at 4°C. Figure 1 As shown, the thiol group of bHb reacts with the vinyl sulfone group of PEG to obtain PEGylated bHb.

[0054] (3) Separation and purification of PEG-bHb: Size exclusion chromatography was used to purify the PEGylated bHb obtained in step (2). A Superdex 200 gel filter column (1.6cm × 60cm) was preferred. The buffer used for equilibration and elution of the chromatography column was 20mM PBS buffer (pH 7.4). The reaction solution from step (2) was loaded onto the chromatography column, and elution was performed at a flow rate of 2mL / min. The elution peak was detected at 280nm. Figure 3 As shown, collect the eluent corresponding to the elution peak indicated by the arrow, remove the unreacted PEG, which is PEG-bHb, and store it in a -80℃ freezer for later use.

[0055] Example 2: Identification of PEG-bHb by gel filtration

[0056] PEG-bHb and bHb were analyzed using an analytical Superdex 200 semi-preparative gel filtration column (1 cm × 30 cm).

[0057] like Figure 4 As shown, both PEG-bHb and bHb exhibited single and symmetrical elution peaks after elution, with peak positions at 12.7 mL and 17.9 mL, respectively. This indicates that both samples possess high purity. Compared to bHb, the elution time of PEG-bHb shifted significantly to the left. This suggests that PEG modification significantly increased the hydration volume of bHb.

[0058] Example 3: SDS-PAGE electrophoresis analysis

[0059] Add an equal volume of electrophoresis buffer to the PEG-bHb solution, place in a boiling water bath for 5 minutes, and analyze the purity of PEG-bHb by SDS-PAGE electrophoresis using a 15% electrophoresis gel (coomassie brilliant blue stained).

[0060] like Figure 5 As shown, bHb (lane 2) exhibits a single electrophoretic band, corresponding to a molecular weight of approximately 16 kDa. This is because bHb (64.0 kDa) is composed of four subunits, which completely depolymerize into a single subunit with a molecular weight of 16.0 kDa during a boiling water bath. Compared to bHb, PEG-bHb (lane 3) shows four additional electrophoretic bands, while the original bands become lighter. This indicates that almost all four subunits of bHb are modified with PEG, and each subunit binds 1–4 PEG molecules, with only a small number of unmodified subunit bands appearing. Therefore, hemoglobin and PEG covalently bind, resulting in PEGylated bHb.

[0061] Example 4: Molecular diameter of PEG-bHb

[0062] The molecular diameter of PEG-bHb was determined by dynamic light scattering. For example... Figure 6 As shown, the molecular diameters of PEG-bHb and bHb are 10.9 nm and 5.4 nm, respectively, and their particle sizes exhibit a symmetrical single-peak distribution. The molecular diameter of PEG-bHb is larger than that of bHb. This indicates that PEG modification significantly increases the molecular diameter of bHb.

[0063] Example 5: Structural Analysis of PEG-bHb

[0064] The circular dichroism spectra of PEG-bHb and bHb in the near-ultraviolet region (260-480 nm) were detected using a circular dichroism spectrometer to characterize the secondary structure and heme region structure of PEG-bHb. Three measurements were taken against a buffer background, and the average value was calculated. The CD value (mdeg) of the samples was also determined at 25℃.

[0065] like Figure 7As shown, the spectrum of PEG-bHb is slightly higher than that of bHb in the region near the L band (around 260 nm). The region near the L band is sensitive to the interaction between heme and its surrounding globins and is affected by ligand interactions (such as oxygen or carbon monoxide). Therefore, PEG modification has a slight effect on the interaction between heme and its surrounding globins. The maximum absorbance of PEG-bHb near the Soret peak (around 420 nm) is slightly higher than that of bHb. The wavelength corresponding to the maximum absorbance of bHb is 420 nm, while the wavelength corresponding to the maximum absorbance of PEG-bHb is 418 nm. The absorbance of PEG-bHb is also slightly higher than that of bHb in other regions. This indicates that PEG modification slightly interferes with the heme microenvironment structure of bHb.

[0066] Example 6: Colloidal osmotic pressure of PEG-bHb

[0067] The colloidal osmotic pressures of PEG-bHb and bHb solutions were measured using a Wescor 4420 colloid osmometer (Wescor, USA) at room temperature. Both PEG-bHb and bHb solutions were thoroughly dialyzed with PBS buffer (pH 7.4), and the protein concentration in both solutions was diluted to 40 mg / mL after dialyzing. The colloid osmotic pressure of the PEG-bHb solution at a protein concentration of 40 mg / mL was measured to be 49.6 mmHg, significantly lower than the colloid osmotic pressure of PEGylated bovine hemoglobin (>200 mmHg) from Enzon Pharmaceuticals at the same protein concentration, but higher than the colloid osmotic pressure of bHb at the same concentration (16.0 mmHg).

[0068] Example 7: UV-Vis Spectroscopic Analysis of PEG-bHb

[0069] like Figure 8 As shown, PEG-bHb and bHb exhibit maximum absorption peaks at 415 nm, 540 nm, and 575 nm, respectively, and the shapes of their spectral absorption curves do not change significantly. This indicates that both PEG-bHb and bHb are in an oxygenated state. At 415 nm, the absorbance values ​​of PEG-bHb and bHb almost overlap, and their peak shapes at 540 nm and 575 nm are very similar.

[0070] Example 8: Oxygen-carrying and releasing function of PEG-bHb

[0071] The oxygen-carrying and releasing capacity of PEG-bHb was determined using a Hemox pulse oximeter from TCS Scientific, USA. 6 mg of PEG-bHb and 6 mg of bHb were added to 4 mL of Hemox buffer, followed by 20 μL of bovine serum albumin and 10 μL of defoamer. The mixture was incubated at 37°C for 5 minutes. The sample was then placed in the sample cell of the pulse oximeter, and the measurement was started after the temperature returned to 37°C.

[0072] like Figure 9 As shown, under physiological oxygen partial pressure conditions (90-100 mmHg), the oxygen-carrying curves of both PEG-bHb and bHb are in a saturated state, indicating that PEG-bHb can effectively carry oxygen. Compared with bHb, the oxygen-carrying curve of PEG-bHb shifts to the left, indicating that the oxygen release capacity of PEG-bHb is adversely affected. 50 The partial pressure of oxygen (i.e., the oxygen partial pressure at which 50% oxygen saturation is achieved) is an important indicator for measuring the oxygen-carrying capacity of an oxygen carrier. 50 The smaller the value, the higher the oxygen affinity of bHb and the lower its oxygen release capacity. Among these, the P value of bHb... 50 The value was 25.6 mmHg, while the P of PEG-bHb was... 50 The value was 10.7 mmHg, indicating that PEG modification disrupted the structural transition of bHb from the tense to the relaxed state to some extent, reducing the P of PEG-bHb. 50 The Hill coefficient of bHb is 2.8, the Hill coefficient of PEG-bHb is 1.9, and the Hill coefficient of PEGylated human hemoglobin from Sangard, Inc. is only about 1.5, indicating that PEG modification only has a slight effect on the synergy among the four subunits of bHb.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A polyethylene glycol-modified bovine hemoglobin oxygen carrier, characterized in that, The bovine hemoglobin surface is modified with methoxy polyethylene glycol ethylene sulfone; Prior to the modification of the bovine hemoglobin with methoxy polyethylene glycol sulfone, the bovine hemoglobin had already been thiolated. The P of the polyethylene glycolated bovine hemoglobin 50 The value is not less than 10 mmHg; The method for preparing the polyethylene glycol-modified bovine hemoglobin oxygen carrier includes: mixing and reacting bovine hemoglobin with methoxy polyethylene glycol vinyl sulfone to obtain polyethylene glycol-modified bovine hemoglobin; wherein the bovine hemoglobin is subjected to thiolation treatment; The method of thiolation is as follows: reacting 2-iminothione with bovine hemoglobin to convert the amino group of bovine hemoglobin into a thiol group; The molar ratio of bovine hemoglobin to 2-iminothion is 1:40; The thiolized bovine hemoglobin is mixed with the methoxy polyethylene glycol sulfone in a molar ratio of 1:20; The molecular weight of the methoxy polyethylene glycol ethylene sulfone is 5 kDa; The PEGylated bovine hemoglobin has a colloidal osmotic pressure of 49.6 mmHg in a colloidal solution with a protein concentration of 40 mg / mL.

2. The polyethylene glycol-modified bovine hemoglobin oxygen carrier according to claim 1, characterized in that, The bovine hemoglobin was dissolved in PBS buffer and then mixed with 2-iminothion dissolved in PBS buffer to obtain thiolated bovine hemoglobin.

3. The polyethylene glycol-modified bovine hemoglobin oxygen carrier according to claim 1, characterized in that, The mixing reaction was terminated by adding glycine.

4. The polyethylene glycol-modified bovine hemoglobin oxygen carrier according to claim 1, characterized in that, The method further includes purifying the crude polyethylene glycolated bovine hemoglobin.

5. The polyethylene glycol-modified bovine hemoglobin oxygen carrier according to claim 4, characterized in that, The purification method is size exclusion chromatography.

6. The use of polyethylene glycol-modified bovine hemoglobin oxygen carrier according to any one of claims 1 to 5 in the preparation of blood substitutes and / or oxygen carriers.

7. The application according to claim 6, characterized in that, The polyethylene glycol-modified bovine hemoglobin oxygen carrier partially or completely replaces red blood cells in carrying or releasing oxygen during blood transfusion.

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