Artificial blood and preparation method thereof

Abstract

The invention relates to artificial blood and a preparation method thereof, and the preparation method comprises the following steps: dissolving glutaraldehyde polymerized hemoglobin, serum albumin and hydroxyethyl starch in a sterile sodium chloride solution, adjusting the pH value of the solution to 7.3-7.5, and filtering to obtain the artificial blood. Compared with the prior art, the preparation method has the advantages that the structural stability of hemoglobin is remarkably improved by adopting glutaraldehyde to polymerize the hemoglobin, the hemoglobin is not easy to decompose into dimers, so that the half-life period of the hemoglobin in blood circulation is prolonged to 24-36 hours from the traditional 2-4 hours, the oxygen carrying capacity of the hemoglobin after glutaraldehyde polymerization is remarkably improved, and the preparation method is simple and convenient to operate. Besides, serum albumin is added into the artificial blood and used for maintaining plasma colloid osmotic pressure, eliminating ascites, inducing diuresis, dehydrating and providing nutrition, and the situation that albumin in blood of a blood transfusion object is sharply diluted in the blood transfusion process, and other adverse effects are caused is avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of artificial blood, and in particular to artificial blood and a preparation method thereof. Background Art

[0002] Blood is an important tissue in the human body. It delivers nutrients and oxygen to various organs of the human body to maintain the normal functioning of the body. When the blood loss exceeds 20% of the total blood volume, life will be threatened. Therefore, blood transfusion is the most effective way to save lives in the case of severe blood loss. However, the amount of blood donated by people often cannot keep up with the amount of blood needed by society.

[0003] In order to cope with the problem of insufficient blood donations, scientists from all over the world have been working on blood substitutes, including artificial blood, platelet substitutes, red blood cell substitutes, etc. The supply of blood substitutes is crucial for performing surgery. For example, red blood cell substitutes can be used as oxygen therapy agents for emergency resuscitation, treatment of ischemic cardiovascular and cerebrovascular diseases or tumor radiosensitization and chemotherapy, etc., which has a strong practical significance. In addition, in the absence of fresh blood and urgent need for blood transfusion, if the patient or family members agree, the use of blood substitutes is also an effective method, especially for people with rare blood types, artificial blood can help them perform surgery in time. However, current artificial blood has problems such as short half-life, easy to cause immune response, and easy to produce nephrotoxicity. Summary of the invention

[0004] The object of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide an artificial blood and a method for preparing the same.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for preparing artificial blood comprises the following steps: dissolving glutaraldehyde polymerized hemoglobin, serum albumin and hydroxyethyl starch in a sterile sodium chloride solution, adjusting the pH of the solution to 7.3-7.5 and filtering to obtain artificial blood.

[0007] Compared with the prior art, the present invention uses glutaraldehyde to polymerize hemoglobin, which significantly improves the structural stability of hemoglobin and makes it less likely to decompose into dimers, thereby extending the half-life of hemoglobin in the blood circulation from the traditional 2-4 hours to 24-36 hours. In addition, the oxygen carrying capacity of hemoglobin after glutaraldehyde polymerization is significantly improved. In addition, the artificial blood of the present invention is added with serum albumin to maintain plasma colloidal osmotic pressure, eliminate ascites, promote diuresis, dehydration, and provide nutrition, thereby avoiding the rapid dilution of albumin concentration in the blood of the transfusion recipient during the transfusion process, causing other adverse effects.

[0008] Furthermore, the concentrations of the glutaraldehyde-polymerized hemoglobin, serum albumin, and hydroxyethyl starch are 50-100 mg / mL, 10-20 mg / mL, and 10-20 mg / mL, respectively.

[0009] Furthermore, the serum albumin is human serum albumin with a molecular weight of 60-70 kDa.

[0010] Furthermore, the glutaraldehyde polymerized hemoglobin is glutaraldehyde polymerized bovine hemoglobin with a molecular weight of 64-800 kDa.

[0011] Furthermore, the preparation method of glutaraldehyde-polymerized bovine hemoglobin includes the following steps: dissolving a freeze-dried bovine hemoglobin sample and glutaraldehyde in a molar ratio of 1:8-12 in a reaction solution, and fully dissolving to obtain a mixed solution; adjusting the pH of the mixed solution to 9.0-9.5 and performing a deoxygenation treatment; in a nitrogen atmosphere, gently stirring the deoxygenated mixed solution at 0-4°C for 1 hour, during which sodium borohydride is added in four batches; then stirring the mixture at 0-4°C for overnight without oxygen to obtain a reaction solution; ultrafiltering, concentrating, and freeze-drying the reaction solution to obtain glutaraldehyde-polymerized bovine hemoglobin; wherein the reaction solution is a mixed solution of sodium chloride and disodium hydrogen phosphate.

[0012] Furthermore, the molar ratio of the sodium borohydride to the freeze-dried bovine hemoglobin sample is 1:9-12.

[0013] Furthermore, the preparation method of the freeze-dried bovine hemoglobin sample includes: taking fresh bovine blood, centrifuging, taking the lower layer of red blood cells for washing, centrifuging, removing the supernatant, breaking the membrane, concentrating, and freeze-drying to obtain the freeze-dried bovine hemoglobin sample.

[0014] Furthermore, the filtration includes sterilizing and filtering the solution using a 0.22 micron filter membrane.

[0015] Furthermore, the artificial blood has an osmotic pressure of 280-330 mmHg, a chloride ion content of 96-106 mmol / L, a sodium ion content of 135-155 mmol / L, and an endotoxin content of less than 0.2 Eu / mL.

[0016] The present invention also provides an artificial blood, which comprises: 50-100 mg / mL of glutaraldehyde-polymerized bovine hemoglobin, 10-20 mg / mL of human serum albumin, and 10-20 mg / mL of hydroxyethyl starch.

[0017] For better understanding and implementation, the present invention is described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the reaction site structural formula of glutaraldehyde polymerization hemoglobin.

[0019] Figure 2 It is a gel electrophoresis diagram, in which 1 is a marker, 2 is pure bovine hemoglobin, and 3-5 are glutaraldehyde-polymerized bovine hemoglobin prepared in Experimental Example 1-3.

[0020] Figure 3 Schematic diagram of the finished product of artificial blood.

[0021] Figure 4 Schematic diagram of the finished product of glutaraldehyde-polymerized bovine hemoglobin. DETAILED DESCRIPTION

[0022] The present invention further studies the problems of artificial blood in the prior art, such as short half-life, easy to cause immune response, and produce adverse effects such as nephrotoxicity, and finds that the hemoglobin (Hb) extracted from natural red blood cells has a high colloidal osmotic pressure in plasma, and the tetramer of the molecule is easily degraded into the form of αβ dimer after infusion, so the retention time of hemoglobin in the blood circulation is short, and the half-life is generally only 2-4h, and the dimer and monomer decomposed by hemoglobin are filtered by the glomerulus and separated from the body fluid circulation, resulting in an increase in protein concentration in the renal tubules, producing strong nephrotoxicity and causing renal dysfunction. In addition, free hemoglobin is easy to lose the regulation of 2,3-DPG in red blood cells, thereby causing an increase in oxygen affinity, making it unable to effectively supply oxygen to tissues. In severe cases, the free hemoglobin tetramer molecule and its decomposition products may also penetrate the vascular endothelial system into organs and tissue interstitium to cause serious side effects. Therefore, hemoglobin extracted from natural red blood cells cannot be used as a blood substitute even when it is highly purified.

[0023] Based on this, the present invention first screens and improves the source and structure of hemoglobin to improve the structural stability and oxygen carrying capacity of hemoglobin: a variety of substances are used to form polymers with hemoglobin respectively, and it is found that adding glutaraldehyde to polymerize hemoglobin can improve the structural stability of the tetramer of the polymer, making it difficult to decompose into dimers, thereby increasing the half-life of hemoglobin from 2-4h to 24-36h, and compared with other hemoglobins, bovine hemoglobin has a significant Bohr effect, and oxygen release to tissues increases at low pH, thereby increasing the oxygen carrying capacity P50 of glutaraldehyde polymerized hemoglobin to 33-38mmHg, which is significantly higher than 25-30mmHg of unpolymerized hemoglobin after in vitro separation, and the oxygen affinity of bovine hemoglobin is not regulated by 2,3-DPG, but by chloride ions present in plasma, so there is no need for modification of 2,3-DPG analogs, and oxygen affinity can be avoided to increase oxygen supply deterioration. In addition, bovine hemoglobin has a wide source and is cheap, which helps to promote the application of bovine blood resources.

[0024] Next, the present invention further studies the ratio of glutaraldehyde to bovine hemoglobin, the concentration of hemoglobin polymer, etc., and finds that within a certain ratio range, the intramolecular polymerization of glutaraldehyde changes the structure of the antigenic determinant cluster of bovine hemoglobin, so that the bovine hemoglobin modified by the polymer has no immunogenicity, is not restricted by blood type, and does not cause severe hemolytic reaction after blood transfusion. It can be used immediately without checking blood type or doing cross-matching test. Therefore, the artificial blood provided by the present invention can be used for emergency treatment, especially large-scale on-site emergency treatment, more simply and quickly, and precious emergency treatment time can be gained for patients. The colloidal osmotic pressure of hemoglobin polymer in plasma is relatively high, and controlling the concentration of hemoglobin solution to below 14g / L, preferably 7g / L, can improve the performance of hemoglobin polymer.

[0025] Furthermore, the present invention explored other components of artificial blood and found that adding 130 / 0.4 hydroxyethyl starch, a blood volume expander, can increase the colloid osmotic pressure of artificial blood and maintain blood pressure. Then, the present invention further adjusted the ratio of hydroxyethyl starch to glutaraldehyde-polymerized hemoglobin, so that artificial blood can reach an oxygen transport capacity index close to that of blood.

[0026] Furthermore, in order to avoid the rapid dilution of albumin concentration in human blood during blood transfusion, the present invention adds a certain proportion of human serum albumin to artificial blood, which is mainly used to maintain plasma colloidal osmotic pressure, eliminate ascites, diuresis, dehydration, and provide nutrition. Human serum albumin and glutaraldehyde-polymerized bovine hemoglobin are combined without side effects, and can be stored in the form of freeze-dried powder with a long shelf life. When artificial blood needs to be used for emergency treatment of blood loss in other animals, especially for pet cats, pet dogs, and endangered animals, serum albumin from the corresponding source can be used to prevent artificial blood from causing animal immune response. Based on the above components, the present invention further adjusts the ratio and concentration between the components to optimize the performance of artificial blood.

[0027] Based on the above research, the present invention provides an artificial blood, comprising 50-100 mg / mL of glutaraldehyde-polymerized bovine hemoglobin, 10-20 mg / mL of human serum albumin, and 10-20 mg / mL of hydroxyethyl starch. The preparation method thereof comprises the following steps: dissolving 5-10 g of glutaraldehyde-polymerized bovine hemoglobin, 1-2 g of human serum albumin, and 1-2 g of hydroxyethyl starch 130 / 0.4 in 100 mL of sterile 0.9% sodium chloride solution, and after complete dissolution, adjusting the pH to 7.4 with sterile 0.1 M sodium hydroxide, and then sterilizing and filtering through a 0.22 micron filter membrane to obtain the artificial blood.

[0028] Wherein, the human serum albumin was purchased from Shenzhen Weiguang Biological Products Co., Ltd., and hydroxyethyl starch was purchased from Aladdin Mall. The preparation method of glutaraldehyde-polymerized bovine hemoglobin includes the following steps:

[0029] Fresh bovine blood is taken, centrifuged, the lower layer of red blood cells is taken for washing, centrifugation, removal of supernatant, membrane breaking, concentration, obtaining a bovine hemoglobin solution, and the bovine hemoglobin solution is freeze-dried to obtain a freeze-dried bovine hemoglobin sample. The freeze-dried bovine hemoglobin sample and glutaraldehyde at a molar ratio of 1:10 are dissolved in a reaction solution, and fully dissolved to obtain a mixed solution; the pH of the mixed solution is adjusted to 9.0-9.5, and a deoxygenation treatment is performed; in a nitrogen atmosphere, the deoxygenated mixed solution is gently stirred at 0-4°C for 1h, during which sodium borohydride is added in four batches (the total molar ratio of sodium borohydride to bovine hemoglobin is 1:10.5); then the reaction is stirred overnight at 0-4°C without oxygen to obtain a reaction solution; the reaction solution is ultrafiltered, concentrated, and freeze-dried to obtain glutaraldehyde-polymerized bovine hemoglobin. The reaction solution includes 100 mmol / L NaCl and 100 mmol / L Na2HPO4, the pH of the reaction solution is 9.5, and the molecular weight of the glutaraldehyde-polymerized hemoglobin is distributed between 64-800 kDa.

[0030] Furthermore, before preparing artificial blood, sterile bottles, medicine spoons, analytical balances, weighing paper and other tools are prepared. The medicine spoons are soaked in 0.5M sodium hydroxide for 1 hour to remove endotoxins, then washed with water until neutral, dried, and sterilized at 120°C for standby use; the analytical balance is wiped and sterilized with 75% alcohol, and the weighing paper is sterilized by irradiation with ultraviolet light for 30-60 minutes.

[0031] The artificial blood provided in this embodiment has a pH of 7.35-7.45, an osmotic pressure of 280-330 mmHg, a chloride ion content of 96-106 mmol / L, a sodium ion content of 135-155 mmol / L, and an endotoxin content of less than 0.2 Eu / mL, which are similar to the transport oxygen carrying index of blood.

[0032] The present invention is further described in detail below in conjunction with specific examples. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified, and the materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0033] Example 1

[0034] This embodiment provides a method for preparing artificial blood, which specifically includes the following steps:

[0035] S1: Weigh a certain amount of freeze-dried bovine hemoglobin sample and glutaraldehyde (the molar ratio of freeze-dried bovine hemoglobin sample to glutaraldehyde is 1:10) and dissolve them in a certain volume of reaction solution (the reaction solution includes 100mmol / L NaCl, 100mmol / LNa2HPO4, and the pH of the reaction solution is 9.5), stir to fully dissolve it to obtain a mixed solution. Use Na2HPO4 to adjust the pH of the mixed solution to 9.0-9.5, and place the mixed solution in a vacuum dryer for deoxygenation, and use a blood gas analyzer to monitor the degree of deoxygenation. After vacuum deoxygenation, fill the mixed solution with nitrogen and place it in an ice water bath (0-4°C) for gentle stirring for 1h. During this period, sodium borohydride is added in four batches, and then continue to react overnight at 4°C without oxygen by stirring to obtain a reaction solution. The reaction solution is ultrafiltered and desalted using a 10kDa ultrafiltration membrane to remove unreacted glutaraldehyde and by-products, and then concentrated and freeze-dried to obtain glutaraldehyde-polymerized bovine hemoglobin. The molar ratio of sodium borohydride to bovine hemoglobin is 1:10.5, the mass ratio of bovine hemoglobin to reaction solution is 1:50, the molecular weight of the glutaraldehyde-polymerized hemoglobin is distributed between 64-800 kDa, and the glutaraldehyde-polymerized hemoglobin includes intramolecularly polymerized glutaraldehyde-polymerized hemoglobin and intermolecularly polymerized glutaraldehyde-polymerized hemoglobin, and the molecular weight of the intramolecularly polymerized glutaraldehyde-polymerized hemoglobin is about 64 kDa.

[0036] S2: In a sterile operating table, 6 g of glutaraldehyde-polymerized bovine hemoglobin, 1 g of human serum albumin, and 2 g of hydroxyethyl starch 130 / 0.4 are weighed using a sterilized weighing tool, and dissolved in 100 mL of a sterile 0.9% sodium chloride solution. After complete dissolution, the solution pH is adjusted to about 7.4 using sterile 0.1 M sodium hydroxide, and then sterilized and filtered through a 0.22 μm filter membrane into a sterile blood bag to obtain artificial blood. The molecular weight of the human serum albumin is about 66 kDa, and the molecular weight of the hydroxyethyl starch is about 130 kDa.

[0037] The artificial blood provided in this embodiment can be prepared by first preparing glutaraldehyde polymerized bovine hemoglobin, and then storing glutaraldehyde polymerized bovine hemoglobin, human serum albumin, and hydroxyethyl starch in a dry powder state, and the shelf life is up to 18 months. When the artificial blood is needed, the artificial blood can be prepared with glutaraldehyde polymerized bovine hemoglobin in a dry powder state, and the artificial blood can be prepared and used immediately, so as to avoid the increase of the content of methemoglobin due to the long preparation time, thereby causing side effects to the body.

[0038] Experimental Example 2

[0039] The method of this embodiment is similar to that of Embodiment 1, except that in this embodiment, the amount of glutaraldehyde-polymerized bovine hemoglobin added in step S2 is 6.5 g, and the amount of hydroxyethyl starch added is 1.5 g.

[0040] Experimental Example 3

[0041] The method of this embodiment is similar to that of Embodiment 1, except that in this embodiment, in step S2, the amount of glutaraldehyde-polymerized bovine hemoglobin added is 7 g, the amount of human serum albumin added is 0.5 g, and the amount of hydroxyethyl starch added is 1.5 g.

[0042] Test Example 1

[0043] The sodium ion content, chloride ion content, colloid osmotic pressure, endotoxin content, oxygen carrying capacity P50, pH value and methemoglobin of the artificial blood prepared in Examples 1-3 were measured. Among them, the sodium ion content and chloride ion content were measured by a biochemical analyzer; the P50 value was the oxygen partial pressure measured when hemoglobin Hb reached 50% oxygen saturation under standard conditions (normal level of whole blood 2,3-DPG, 37°C, pH7.40) (the average oxygen partial pressure of normal people in the plain is 26±2 mmHg), which was measured by a blood oxygen analyzer; the osmotic pressure was measured by an osmometer; the methemoglobin was measured by a colorimetric method, the principle of which is that the methemoglobin forming agent can convert hemoglobin in red blood cells into methemoglobin, and methemoglobin can quickly combine with cyanide ions to form a cyanide methemoglobin complex, which specifically includes the following steps:

[0044] 1. Prepare 0.1M PBS buffer at pH 6.6, 20% sodium ferrocyanide solution and 10% neutral solution (20% NaCN aqueous solution + 20% glacial acetic acid, prepared within 1 hour before the experiment).

[0045] 2. Take two test tubes with caps as standard tubes and measuring tubes. Add 0.1mL Hb solution, 0.1mL potassium ferricyanide solution and 9.8mL PBS buffer to the standard tube; add 0.1mL Hb solution and 9.9mL PBS buffer to the measuring tube, mix well, and let stand at room temperature for 2 minutes. Then use PBS solution as reference and perform colorimetry at a wavelength of 630nm to obtain the absorbance A1 of the measuring tube and the absorbance A2 of the standard tube.

[0046] 3. Add 1 drop of neutral KCN solution to the measuring tube and the standard tube respectively, mix well, let stand at room temperature for 2 minutes, and perform colorimetry at 630 nm to obtain the absorbance A3 of the measuring tube and the absorbance A4 of the standard tube.

[0047] 4. Calculate the methemoglobin content according to the following formula. Note that three samples should be measured at the same time and the average value should be taken as the test result: MetHb% = (A1-A2) / (A3-A4)*100%

[0048] Referring to Table 1, the P50 of the artificial blood provided by the present invention is 31-34 mmHg, which is significantly higher than the P50 of unpolymerized hemoglobin after ex vivo treatment (25-30 mmHg). In addition, the P50 of glutaraldehyde-polymerized hemoglobin measured by the present invention under the same concentration and conditions is about 33-38 mmHg.

[0049] Table 1 Biochemical indicators of artificial blood.

[0050]

[0051] Test Example 2

[0052] The glutaraldehyde-polymerized hemoglobin and the pure bovine hemoglobin in Examples 1 to 3 were subjected to SDS-PAGE gel electrophoresis, comprising the following steps:

[0053] 1. Clean the glass plate: Use detergent to scrub the glass plate, then rinse with tap water and distilled water and dry it.

[0054] 2. Glue pouring: Align the glass plates and put them into the clamps to clamp them tightly, then clamp them vertically on the rack to prepare for glue pouring; prepare 12% separation gel, add TEMED and shake well immediately before pouring; after the separation gel solidifies, prepare 5% concentrated gel, add TEMED and shake well immediately before pouring; after the concentrated gel is filled, insert the comb into the concentrated gel, and after the concentrated gel solidifies, pinch the two sides of the comb with both hands and gently pull it out vertically upwards. Put the gel into the electrophoresis tank.

[0055] 3. Sample loading: Measure the protein content and calculate the volume of the solution containing 50ng of protein as the sample loading amount. Add 5× SDS loading buffer (containing β-mercaptoethanol) to the sample to a final concentration of 1×. Boil the sample in boiling water for 5 minutes before loading to denature the protein. Add enough electrophoresis solution and start preparing for sample loading.

[0056] 4. Electrophoresis: The electrophoresis time is generally 1.5 hours, the voltage is 80V, 30 minutes, and then 120V, 50 minutes. Electrophoresis can be terminated when bromophenol blue just runs out, and transfer to the membrane is performed.

[0057] 5. After transfer, stain the membrane with bromophenol blue staining solution on a shaker for 40 minutes, pour out the staining solution, add decolorizing solution and decolorize on a shaker until the background color is completely removed and the protein bands appear.

[0058] Electrophoresis results refer to Figure 2 .

[0059] In summary, compared with the prior art, the present invention has the following advantages and effects:

[0060] (1) The glutaraldehyde-polymerized hemoglobin used in the present invention can be used as an oxygen carrier. In hemoglobin, the amino and thiol groups of the side chains of amino acid residues such as lysine, arginine, asparagine, glutamine, and the terminal amino groups of the α chain and β chain are all reactive groups of hemoglobin reaction, which are the basis for the cross-linking reaction and cross-reaction of hemoglobin. The differences in these sites of hemoglobin from different sources can cause different changes in the immunological properties of hemoglobin after polymerization, and the modification of these sites may also cause the disappearance of the original immunogenicity of natural hemoglobin or the generation of new immunogenicity. The antigenic determinant of the natural bovine hemoglobin used in the present invention disappears after the glutaraldehyde polymerization reaction, and the antigenic determinant is wrapped in the protein and cannot be exposed, so that the glutaraldehyde-polymerized hemoglobin has no immunogenicity. When it is used for blood transfusion, no serious hemolytic reaction will occur, and it can be used immediately without checking the blood type or doing a cross-matching test.

[0061] (2) The present invention significantly improves the structural stability of hemoglobin by polymerizing hemoglobin with glutaraldehyde, making it less likely to decompose into dimers, thereby extending the half-life of hemoglobin in the blood circulation from the traditional 2-4 hours to 24-36 hours.

[0062] (3) The present invention uses bovine hemoglobin as a raw material and utilizes its significant Bohr effect to increase the oxygen release of polymerized hemoglobin to tissues at low pH. The oxygen carrying capacity P50 is higher than that of unpolymerized hemoglobin, thereby improving the oxygen carrying capacity of artificial blood.

[0063] (4) The artificial blood provided by the present invention is mainly used for emergency treatment of human blood loss. Therefore, human serum albumin is used in combination with other components. The main functions of human serum albumin are to maintain plasma colloidal osmotic pressure, eliminate ascites, promote diuresis, dehydrate, and provide nutrition. There is no side effect when it is combined with glutaraldehyde-polymerized bovine hemoglobin. The addition of human albumin can prevent the albumin concentration in human blood from being rapidly diluted during blood transfusion, causing other adverse effects such as edema and decreased immunity in patients.

[0064] (5) The present invention can replace the source of serum albumin in artificial blood with serum albumin from animals that need to be rescued, and has a wide range of application scenarios, avoiding the inability to rescue animals in time due to lack of animal blood banks, especially the lives of endangered animals and pets.

[0065] (6) Artificial blood can be stored in the form of freeze-dried powder with a long shelf life. It only needs to be prepared and used immediately, avoiding the problem of increased methemoglobin content due to long preparation time, thus ensuring the safety and effectiveness of use.

[0066] (7) The preparation method provided by the present invention is simple and easy to operate. By precisely controlling the reaction conditions and ratios, artificial blood that meets the requirements can be efficiently prepared. In addition, strict sterilization and aseptic operations are adopted during the preparation process to ensure the sterility and safety of the artificial blood.

[0067] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, and the present invention is also intended to include these modifications and modifications.

Claims

1. A method for preparing artificial blood, characterized in that: The method comprises the following steps: dissolving glutaraldehyde polymerized hemoglobin, serum albumin and hydroxyethyl starch in a sterile sodium chloride solution, adjusting the pH of the solution to 7.3-7.5 and filtering the solution to obtain artificial blood.

2. The method according to claim 1, characterized in that: The concentrations of the glutaraldehyde-polymerized hemoglobin, serum albumin and hydroxyethyl starch are 50-100 mg / mL, 10-20 mg / mL and 10-20 mg / mL respectively.

3. The method according to claim 2, characterized in that: The serum albumin is human serum albumin, and has a molecular weight of 60-70 kDa.

4. The method according to claim 3, characterized in that: The glutaraldehyde polymerized hemoglobin is glutaraldehyde polymerized bovine hemoglobin with a molecular weight of 64-800 kDa.

5. The method according to claim 4, characterized in that: The preparation method of glutaraldehyde-polymerized bovine hemoglobin comprises the following steps: dissolving a freeze-dried bovine hemoglobin sample and glutaraldehyde at a molar ratio of 1:8-12 in a reaction solution to obtain a mixed solution; adjusting the pH of the mixed solution to 9.0-9.5 and performing a deoxygenation treatment; in a nitrogen atmosphere, gently stirring the deoxygenated mixed solution at 0-4°C for 1 hour, during which sodium borohydride is added in four batches; then stirring the mixed solution at 0-4°C for overnight reaction without oxygen to obtain a reaction solution; ultrafiltration, concentration and freeze-drying the reaction solution to obtain glutaraldehyde-polymerized bovine hemoglobin; wherein the reaction solution is a mixed solution of sodium chloride and disodium hydrogen phosphate.

6. The method according to claim 5, characterized in that: The molar ratio of the sodium borohydride to the freeze-dried bovine hemoglobin sample is 1:9-12.

7. The method according to claim 6, characterized in that: The preparation method of the freeze-dried bovine hemoglobin sample comprises: taking fresh bovine blood, centrifuging, taking the lower layer of red blood cells for washing, centrifuging, removing the supernatant, breaking the membrane, concentrating, and freeze-drying to obtain the freeze-dried bovine hemoglobin sample.

8. The method according to claim 7, characterized in that: The filtration includes sterilizing the solution using a 0.22 micron filter membrane.

9. The method according to claim 8, characterized in that: The artificial blood has an osmotic pressure of 280-330 mmHg, a chloride ion content of 96-106 mmol / L, a sodium ion content of 135-155 mmol / L, and an endotoxin content of less than 0.2 Eu / mL.

10. An artificial blood, characterized in that: include: 50-100 mg / mL glutaraldehyde polymerized bovine hemoglobin, 10-20 mg / mL human serum albumin, 10-20 mg / mL hydroxyethyl starch.

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