CN114617957B - Hydroxyethyl starch hemoglobin conjugate, and preparation method and application thereof - Google Patents
Hydroxyethyl starch hemoglobin conjugate, and preparation method and application thereof Download PDFInfo
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- CN114617957B CN114617957B CN202210240060.5A CN202210240060A CN114617957B CN 114617957 B CN114617957 B CN 114617957B CN 202210240060 A CN202210240060 A CN 202210240060A CN 114617957 B CN114617957 B CN 114617957B
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/41—Porphyrin- or corrin-ring-containing peptides
- A61K38/42—Haemoglobins; Myoglobins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/06—Antianaemics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
- C07K14/805—Haemoglobins; Myoglobins
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- General Health & Medical Sciences (AREA)
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- Engineering & Computer Science (AREA)
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- Pharmacology & Pharmacy (AREA)
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- Animal Behavior & Ethology (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- Gastroenterology & Hepatology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Diabetes (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract
The invention discloses a hydroxyethyl starch hemoglobin conjugate, and a preparation method and application thereof. Hydroxyethyl starch hemoglobin conjugate is obtained by hydroxyethyl starch conjugated hemoglobin. The conjugate provided by the invention can effectively improve the colloid osmotic pressure in blood vessels, thereby effectively increasing and maintaining the blood volume in the blood vessels and promoting the tissue microcirculation during hemorrhagic shock. The hemoglobin in the conjugate can combine, transport and release oxygen to treat tissue hypoxia caused by hemorrhagic shock and severe anemia, and maintain normal metabolism of cells in the body. The hydroxyethyl starch hemoglobin conjugate provided by the invention can effectively solve the problems that tissue hypoxia needs to be relieved and blood volume needs to be enlarged when hemorrhagic shock is clinically treated, and can be used as a plasma dilatant to effectively relieve tissue hypoxia and blood volume reduction caused by the hemorrhagic shock.
Description
Technical Field
The invention relates to the technical field of blood substitutes, in particular to a hemoglobin oxygen carrier blood substitute-hydroxyethyl starch hemoglobin conjugate and a preparation method and application thereof.
Background
Acute massive blood loss can lead to reduced blood circulation in the body, thereby inducing hypovolemic shock. Hypovolemic shock caused by such massive blood loss is called hemorrhagic shock. Is commonly found in bleeding caused by trauma, peptic ulcer bleeding, esophageal varices, bleeding caused by gynecological diseases and the like. Hemorrhagic shock can lead to hypoxia, anaerobic cellular metabolism and hypovolemia, affecting the normal physiological functions and microcirculation (blood circulation between arterioles and venules) of the body. The main treatment means of clinical hemorrhagic shock is blood volume supplementation, which is mainly realized by infusion of whole blood, blood plasma, concentrated red blood cells, blood substitutes and/or fluid supplements (such as infusion of ringer's acetate solution), etc.
Blood transfusion (e.g., whole blood, plasma, concentrated red blood cells) can deliver oxygen to tissues, increase blood volume, promote microcirculation, maintain normal metabolism of cells in the body, and is an effective method for relieving hemorrhagic shock. However, transfusion has some limitations such as the need for cross-matching before transfusion, the risk of transmitting pathogen diseases (such as AIDS and hepatitis C) and eliciting allergic reactions, and the problems of the above blood product supply shortages. In order to solve the problem of blood product supply shortage, blood substitutes have been developed.
Blood substitutes broadly include blood plasma substitutes, platelet substitutes, and red blood cell substitutes. In the case of hemorrhagic shock, in order to rapidly supplement blood volume, a blood plasma substitute is an artificial product for increasing and maintaining blood plasma volume, and after increasing blood plasma volume, the blood plasma volume can promote microcirculation, and has been widely used in acute blood transfusion links such as serious blood loss, apheresis, stable blood circulation, blood dilution and the like, and also called as a blood plasma volume expander. The plasma expander commonly used in clinic at present comprises a crystal solution and a colloid solution. The crystal solution, such as isotonic normal saline, has no antigenicity, no bacteria and no pyrogen, but is isotonic with the body fluid environment, can freely pass through various membrane systems in the body, cannot effectively maintain the osmotic pressure in the blood vessel, and cannot maintain the blood volume in the blood vessel. Colloidal solutions such as dextran, hydroxyethyl starch, human serum albumin, oxidized gelatin, povidone, etc., have higher osmotic pressure than the body fluid environment and are not easily permeable to membranes, and can effectively maintain the blood volume in blood vessels compared with the crystalline solution, thereby promoting tissue microcirculation during hemorrhagic shock and maintaining normal metabolism of cells in the body.
Because hemorrhagic shock is often accompanied by tissue hypoxia, only infusion of a plasma expander can only increase blood volume, and the problems of tissue hypoxia and anaerobic metabolism of cells cannot be solved. In order to treat hemorrhagic shock, there is an urgent need for a product that can both deliver oxygen to tissue and expand blood volume.
Disclosure of Invention
The invention aims at overcoming the technical defects in the prior art, and in a first aspect, the invention provides a hydroxyethyl starch hemoglobin conjugate which can effectively increase blood volume and can deliver oxygen to tissues, wherein the hydroxyethyl starch hemoglobin conjugate is obtained by coupling hemoglobin with hydroxyethyl starch, and the molecular weight of the conjugate is more than 200kDa; preferably, the hemoglobin in the conjugate is of tetrameric structure; more preferably, the hemoglobin secondary structure of the conjugate is intact; and the structure between heme and globin in hemoglobin is not changed.
The spectrum has characteristic peaks at 260nm, 285nm and Soret bands in a circular dichroism spectrum; alternatively, it has three characteristic peaks at 410nm, 540nm and 576nm in the ultraviolet-visible spectrum, preferably it has no peak at 630 nm; alternatively to this, the method may comprise,3300cm of the product in an infrared spectrum -1 、2925cm -1 、2851cm -1 、1650cm -1 And 1540cm -1 With characteristic peaks, preferably at 1540cm -1 Point and 1650cm -1 The characteristic peak intensity at the site is stronger than that of hemoglobin.
Hydroxyethyl starch has a molecular weight of 100-300kDa, preferably 130-200kDa; the hemoglobin is selected from human hemoglobin, bovine hemoglobin or porcine hemoglobin, more preferably the conjugate is bHb-HES130 and/or bHb-HES200.
P of conjugate 50 The value (oxygen partial pressure at 50% oxygen saturation) is 10mmHg to 20mmHg.
In a second aspect, the present invention provides a method for preparing the above hydroxyethyl starch hemoglobin conjugate, comprising the steps of oxidizing hydroxyethyl starch (HES), coupling hemoglobin (Hb) to the oxidized hydroxyethyl starch, and the like.
Hydroxyethyl starch was oxidized with sodium periodate.
Hb, oxidized HES and NaCNBH upon coupling 3 Mix, and Hb: oxidized HES: naCNBH 3 The molar ratio of (2) is 4:3:300.
In a third aspect, the present invention provides the use of the above hydroxyethyl starch hemoglobin conjugate in the preparation of a plasma expander; preferably, the plasma expander has a gel osmotic pressure of 5-30mmHg (preferably 7.5-27.5 mmHg) at a hemoglobin concentration of 5-25 mg/mL.
In a fourth aspect, the present invention provides the use of the above-described hydroxyethyl starch hemoglobin conjugate in the preparation of a medicament for the treatment of hemorrhagic shock (blood loss caused by trauma, peptic ulcer, esophageal varices, gynaecological and obstetrical diseases, perioperative) or severe anemia; preferably, the erythrocyte stiffness index is 5-6 (preferably 5.10-5.80) after the drug is mixed with blood.
The plasma expander or the medicine is applied to blood transfusion processes (such as blood loss, apheresis, stable blood circulation, diluted blood and other acute blood transfusion links), and partial or total replacement of plasma plays a role in increasing blood volume, and partial or total replacement of red blood cells plays a role in carrying oxygen and releasing oxygen, and the oxygen partial pressure P when the oxygen saturation is 50 percent 50 The erythrocyte rigidity index is 5-6 (preferably 5.10-5.80) at 10-20mmHg, and the colloid osmotic pressure is 5-30mmHg when the hemoglobin concentration is 5-25 mg/mL.
The hydroxyethyl starch (HES) hemoglobin (Hb) conjugate provided by the invention is a macromolecular substance formed by coupling HES and Hb through chemical bonds, the osmotic pressure of the HES and Hb conjugate is effectively improved compared with that of HES and Hb, the osmotic pressure in blood vessels can be effectively maintained, and the blood volume in the blood vessels can be effectively increased and maintained, so that tissue microcirculation in hemorrhagic shock is promoted, and normal metabolism of cells in vivo is ensured. Hb in the conjugate is capable of binding, transporting and releasing oxygen to treat hypoxia caused by hemorrhagic shock and maintain normal metabolism of cells in the body. The Hb source in the conjugate can be human, bovine or porcine, wherein bovine hemoglobin (bHb) and porcine hemoglobin are sufficient in storage, controllable in quality, high in homology with human Hb and good in safety. HES130 and HES200 are selected, and have proper molecular weight and low side effects. The hydroxyethyl starch hemoglobin conjugate provided by the invention can effectively solve the problems that tissue hypoxia needs to be relieved and blood volume needs to be enlarged when hemorrhagic shock is clinically treated, can effectively relieve the hemorrhagic shock when being used as a plasma dilatant, and is a new development thought of blood substitutes.
Drawings
FIG. 1 is a schematic illustration of a reaction for preparing hydroxyethyl starch hemoglobin conjugates of the present invention;
FIG. 2 shows SDS-PAGE electrophoresis of bHb-HES of example 1 and example 2, wherein: lane 1 and lane 5 are markers of different molecular weights, lane 2 is bHb, lane 3 is bHb-HES130, and lane 4 is bHb-HES200;
FIG. 3 shows the circular dichroism spectra of bHb-HES of example 1 and example 2;
FIG. 4 is a graph showing the ultraviolet-visible spectrum of bHb-HES of example 1 and example 2;
FIG. 5 shows Fourier transform infrared spectra of bHb-HES of example 1 and example 2;
FIG. 6 shows the colloid osmotic pressure curves of bHb-HES of example 1 and example 2;
FIG. 7 is a bar graph of the erythrocyte stiffness index of bHb-HES of examples 1 and 2.
Detailed Description
Hydroxyethyl starch (HES) is a polymeric complex formed by hydroxyethylating the glucose ring of amylopectin in corn or potato, a highly branched semisynthetic amylopectin. The natural starch is unstable in property and is easy to hydrolyze by endogenous amylase, can not be used as a blood plasma substitute for infusion into a human body, can delay the decomposition and metabolism of starch in blood after hydroxyethylation, remarkably prolongs the residence time of the starch in blood vessels, is an effective blood plasma expander in clinical HES colloidal solution, and has wide application in the aspect of clinical treatment of hypovolemia. However, HES as a plasma expander can have side effects such as allergic reactions, bleeding, and platelet damage when used. The side effects of HES depend primarily on the molecular weight of HES, e.g., HES450 (molecular weight 450 kDa) and HES700 (molecular weight 700 kDa) can lead to bleeding complications and itching; HES70 (70 kDa molecular weight) has low capacity for volume expansion and is rapidly metabolized by the kidneys; while the infusion of HES130 (130 kDa molecular weight) and HES200 (200 kDa molecular weight) as a plasma expander cannot solve the tissue hypoxia caused by hemorrhagic shock, even the concentration of Hb in blood is reduced due to dilution of blood by infusion, which aggravates the tissue hypoxia. Thus, in the treatment of hemorrhagic shock, in addition to the infusion of plasma expanders for expanding blood volume, the infusion of whole blood, concentrated red blood cells, or red blood cell substitutes for compensating for oxygen content in blood is needed to alleviate tissue hypoxia.
Whole blood, concentrated red blood cells or red blood cell substitutes all contain Hemoglobin (Hb) which can bind, transport and release oxygen as an oxygen carrier to alleviate tissue hypoxia caused by hemorrhagic shock. However, since hemoglobin is a protein having a tetrameric structure, the molecule is relatively small, and the tetrameric structure is easily dissociated, and thus, it has renal toxicity after dissociation, and thus, it cannot be directly infused into blood.
The present invention couples HES to Hb via a chemical bond to form a conjugate (Hb is selected from bovine hemoglobin, as represented by the structure bHb-HES). The conjugate can effectively and rapidly increase osmotic pressure compared with HES or bHb used alone and HES+ bHb used in combination, so as to keep infused liquid in blood vessels, expand and maintain blood volume. In addition, the hemoglobin in the conjugate still exists in a tetramer structure, is not dissociated, and can play the functions of combining, conveying and releasing oxygen to relieve tissue hypoxia. The Hb source in the conjugate can be human, bovine or porcine, wherein bovine hemoglobin (bHb) and porcine hemoglobin are sufficient in storage, controllable in quality, high in homology with human hemoglobin and good in safety. HES130 or HES200 are selected for the HES130 or HES200 in the conjugate because HES130 and HES200 have a suitable molecular weight and less side effects. HES has too low molecular weight to maintain osmotic pressure in vivo, too high molecular weight is metabolized slowly in vivo, and has large particle size, and can be phagocytized as foreign matter by reticuloendothelial system to fail to function.
According to the invention, bHb is coupled with HES130 and HES200 respectively to prepare two bHb-HES conjugates (respectively represented by bHb-HES130 and bHb-HES 200). The structure of the two conjugates (test three and test four), the structure of heme (test two) and the oxygen carrying performance (test five) are tested through experiments, so that the effectiveness of the conjugates as oxygen carriers is confirmed; the effectiveness as a plasma expander was also confirmed by measuring COP of these two conjugates (test six) and viscosity after mixing with whole blood (test seven); the effect of the two conjugates on the morphology of erythrocytes (test seven), hemolysis (test eight) and platelet aggregation (test nine) was also determined, confirming their safety as intravenous infusion products. In conclusion, the bHb-HES conjugate provided by the invention has the effects of combining, conveying and releasing oxygen, can expand and maintain blood volume, can simultaneously solve the problems of tissue hypoxia and blood volume reduction caused by hemorrhagic shock, and can be used as an oxygen carrier and a plasma dilatant to effectively relieve hemorrhagic shock.
Meanwhile, the conjugate has the oxygen carrying/releasing function, and can be used for treating severe anemia (clinical severe anemia with the concentration of hemoglobin less than 30g/L in peripheral blood, severe anemia with the concentration of hemoglobin between 30 and 60g/L, moderate anemia with the concentration of hemoglobin between 60 and 90g/L, and mild anemia with the concentration of hemoglobin more than 90g/L and lower than a normal value), for example, ischemia and hypoxia of various tissues and organs can be caused by anemia caused by severe thalassemia, tumors and the like, so that other diseases can be caused, blood transfusion treatment is needed when the anemia is severe, and the conjugate can be used as an oxygen carrier to relieve ischemia and hypoxia of various tissues and organs caused by anemia.
The present invention will be described more specifically with reference to the following examples, which are not intended to limit the present invention in any way.
Example 1 preparation of bHb-HES130 conjugate
The reaction process for preparing the hydroxyethyl starch hemoglobin conjugate of this example is schematically shown in fig. 1, and specifically comprises: HES130 was dispersed in 20mM sodium acetate buffer (pH 5.6) to give a HES solution having a concentration of 50mg/mL for HES130, then sodium periodate (the final concentration of sodium periodate in the reaction system was 200 mM) was added to oxidize HES130, the reaction system was incubated in the dark at room temperature for 30 minutes, then excess ethylene glycol was added to terminate the reaction, and then the reaction system was packed in a dialysis bag (dialysis bag cut-off molecular weight 7000 kDa) and dialyzed in PBS (pH 7.4) buffer until no sodium periodate was present in the dialysate (PBS buffer). Adding bovine hemoglobin and NaCNBH into the dialyzed reaction system 3 So that bHb: oxidized HES130: naCNBH 3 Incubated overnight at 4:3: 300,4 ℃to allow bHb to couple with oxidized HES130 and excess glycine was added to terminate the reaction. The reaction system was loaded onto a Superdex 200 column (2.6 cm. Times. 60cm,GE Healthcare, U.S.) and the Superdex 200 column was equilibrated and eluted with PBS buffer (pH 7.4) using a flow rate of 2mL/min, and the eluate of the absorption peak of bHb-HES130 component was collected and concentrated by ultrafiltration to give a solution containing bHb-HES130 conjugate (abbreviated as bHb-HES 130).
Example 2 preparation of bHb-HES200 conjugate
As in example 1, except that HES130 in example 1 was replaced with HES200, a solution containing bHb-HES200 conjugate (abbreviated as bHb-HES 200) was prepared.
Run one, SDS-PAGE electrophoretic analysis
SDS-PAGE analysis was performed on the PB solution of bHb, the bHb-HES 130-containing solution obtained in example 1 and the bHb-HES 200-containing solution obtained in example 2, using a 12% polyacrylamide gel (5% (v/v) beta-mercaptoethanol) under reducing conditions, the gel was stained with Coomassie Brilliant blue R-250, and the results are shown in FIG. 2 (wherein bands 1-5 are: molecular weight markers, bHb-HES130, bHb-HES200, molecular weight markers, respectively).
FIG. 2 shows that bHb (band 2) is a single band, located at a molecular weight of 16kDa, illustrating dissociation of the tetrameric structure of bHb (molecular weight 64 kDa) into four subunits (molecular weight 16 kDa) under electrophoretic conditions. While bHb-HES130 (lane 3) and bHb-HES200 (lane 4) each exhibit lower mobility than bHb (the closer the lane is to the top representing its mobility in fig. 2, the lower its mobility), the corresponding molecular weight is over 200kDa, indicating that all four globulins of bHb bind to the larger HES and that the tetrameric structure of bHb is retained.
Analysis of spectrum of two colors of test and circle
Typically, after coupling hemoglobin with other substances, interactions between heme and globin in the hemoglobin structure are altered, and the secondary structural integrity of hemoglobin may be compromised, which affects the oxygen carrying capacity of hemoglobin.
The test was performed by analyzing the PB solution of bHb, the bHb-HES 130-containing solution obtained in example 1 and the bHb-HES 200-containing solution obtained in example 2 by circular dichroism (band width: 2nm; wavelength range: 250-190nm; sample cell size: 1 cm) to characterize whether the interaction between heme and globin in hemoglobin in the conjugate was altered and the integrity of the secondary structure of hemoglobin, and the results are shown in FIG. 3.
FIG. 3 shows that bHb-HES130 and bHb-HES200 have characteristic peaks with hemoglobin stock (bHb) at 260nm, 285nm and Soret bands (Soret bands refer to strong absorption peak positions of porphyrin compounds in the 420nm range of the ultraviolet visible region, namely, the highest peak in FIG. 3), indicating that interaction between heme and globin in hemoglobin is not changed during coupling of HES and bHb of example 1 and example 2, and the integrity of secondary structure of hemoglobin is maintained, and the conjugate has oxygen carrying capacity.
Test three, ultraviolet-visible spectral analysis
The PB solution of bHb, the bHb-HES 130-containing solution obtained in example 1 and the bHb-HES 200-containing solution obtained in example 2 were analyzed by a spectrophotometer in the ultraviolet-visible spectrum at 220-650nm, and the chemical composition and structural characteristics thereof were analyzed, and the results are shown in FIG. 4.
FIG. 4 shows that the spectra of bHb at 410nm, 540nm and 576nm, bHb-HES130 and bHb-HES200 almost overlap bHb, and characteristic peaks also appear at these three positions, indicating that HES successfully couples to hemoglobin, the structure of hemoglobin in the conjugate of the invention is unchanged, indicating that the coupling process does not destroy the chemical structure of bHb and the structural features of both are not destroyed, bHb-HES has oxygen carrying capacity. These three characteristic peaks also demonstrate that both bHb-HES130 and bHb-HES200 exist in a fully oxygenated form (the form of oxygenated hemoglobin formed by the combination of hemoglobin in the conjugate with oxygen), indicating that bHb-HES130 and bHb-HES200 have oxygen carrying functions.
In addition, the oxygen-carrying and oxygen-releasing functions of hemoglobin are realized by the dynamic process of combining and releasing ferrous ions and oxygen contained in the hemoglobin. When hemoglobin undergoes autoxidation, ferrous ions are oxidized to ferric ions, forming methemoglobin. Methemoglobin cannot be stably combined with oxygen and loses the oxygen carrying function. No peak at 630nm is seen in FIG. 4, indicating that the absence of methemoglobin in bHb-HES130 and bHb-HES200 suggests that the hemoglobin in the conjugate is not autooxidized and still has oxygen carrying capacity.
Test four, fourier transform Infrared Spectrometry
PB solution of bHb, bHb-HES 130-containing solution obtained in example 1 and bHb-HES 200-containing solution obtained in example 2 were measured by infrared spectrometer at 2000cm -1 -500cm -1 Analyzing the chemical composition and structural characteristics of the material,the results are shown in FIG. 5.
FIG. 5 shows that the FT-IR spectrum of bHb is at 3300cm -1 (N-H stretching), 1650cm -1 (c=o stretch) and 1540cm -1 Characteristic peaks appear at (N-H wobble). The spectral characteristic peaks of HES130 and HES200 are 3300cm respectively -1 (O-H stretching), 2925cm -1 (-CH 2 Asymmetric stretching) and 2851cm -1 (-CH 2 Symmetrically stretched), of which 2925cm -1 And 2851cm -1 The peak at this point is a characteristic peak of hydroxyethyl in HES. The spectral characteristic peaks of bHb-HES130 and bHb-HES200 are 3300cm respectively -1 、2925cm -1 、2851cm -1 、1650cm -1 And 1540cm -1 The presence of both HES and bHb characteristic groups in bHb-HES130 and bHb-HES200 is shown to mean that the conjugate has both hemoglobin and HES structures, and that the hemoglobin structure is unchanged.
Furthermore, bHb-HES130 and bHb-HES200 are at 1540cm -1 The intensity at this point is stronger than bHb, which is the result of the characteristic peak of the imide group formed by the coupling between HES and bHb being superimposed on the characteristic peak at bHb. bHb-HES130 and bHb-HES200 at 1650cm -1 The intensities at these sites were also significantly higher than bHb, which is the result of the superposition of the characteristic peaks at these sites for HES and bHb in the conjugate, indicating that bHb-HES130 and bHb-HES200 still maintain the classical helical structure of bHb.
Test five, oxygen affinity assay
1.5mL of the PB solution of bHb (PB: pH7.4,0.2M), the solution containing bHb-HES130 obtained in example 1 and the solution containing bHb-HES200 obtained in example 2 were respectively dispersed in 4mL of a Hemoscan buffer (available from Beijing Kai Biotechnology Co., ltd.) of pH7.4 at 37℃and mixed uniformly as a reaction system in which the final concentration of hemoglobin was 1.5mg/mL. The temperature of the reaction system was controlled to 37℃and the oxygen balance curve of each reaction system was measured by using a Hemox analyzer from TCS Scientific, USA, and P was calculated from the oxygen balance curve 50 Value (oxygen partial pressure at 50% oxygen saturation) and acid-base sensitivity index si= (P at ph 7.2) according to the formula 50 P of value-pH 7.6 50 value)/P of pH7.4 50 Value x 100% calculation gave an acid-base sensitivity index, and the results are shown in table 1.
TABLE 1P of bHb-HES 50 Value of
P of Hb-HES130 and bHb-HES200 50 The values at physiological pH (7.4) were 15.12mmHg and 13.89mmHg, respectively, indicating that they had the capacity to carry oxygen and release oxygen. P (P) 50 The value is the corresponding partial pressure of oxygen, P, when the oxygen saturation of the hemoglobin reaches 50 percent 50 The lower the value, the higher the oxygen affinity. P of bHb-HES130 and bHb-HES200 50 The values are lower than bHb (30.16 mmHg) and the difference is obvious, so that on one hand, the affinity of the hemoglobin to oxygen after the coupling of HES is improved, the hemoglobin is more suitable for oxygen supply of ischemic tissues, and on the other hand, the oxygen release capacity of the hemoglobin after the coupling of HES is weakened, the oxygen can be prevented from being released in advance before the coupling agent reaches the aerobic tissues, and the bioavailability is higher.
In addition, CO in blood at different parts of the body 2 Is different in concentration of CO 2 The concentration of (2) will change the pH of the blood so that the pH of the blood varies between 7.2 and 7.6, and hemoglobin and its substitutes need to have oxygen carrying and releasing capabilities within this pH range, and the variation should not be excessive. The acid-base sensitivity index SI is used to evaluate the degree of sensitivity of hemoglobin and its substitutes to in vivo acid-base. As can be seen from the results of Table 1, the acid-base sensitivity index SI of Hb-HES130 and bHb-HES200 is comparable to bHb, indicating that Hb-HES130 and bHb-HES200 still have oxygen carrying and releasing capabilities and P when the blood pH is varied between 7.2 and 7.6 50 The value is not changed greatly, which is equivalent to bHb; the conjugate provided by the invention can exert the oxygen-carrying and oxygen-releasing functions at different parts in vivo.
Test six, gel osmotic pressure
Substances for regulating the osmotic pressure of the blood plasma in vivo are mainly albumin, but the content of the albumin in the body is reduced in a blood loss state, the osmotic pressure of the blood plasma cannot be effectively maintained, edema can be caused, kidney diseases, liver diseases and the like can be caused, so that the osmotic pressure of the blood plasma expanding agent is increased to be important for maintaining the osmotic pressure of the blood plasma. The osmotic pressure of the fluid infused into the blood vessel, which reflects the ability of the plasma expander to expand and maintain blood volume, is important to maintain the water balance inside and outside the blood vessel.
The PB solution of bHb, the colloid solution of bHb +HES130 blend (bHb with HES130 simply mixed), the colloid solution of bHb +HES200 blend (bHb with HES200 simply mixed), the colloid osmotic pressure (Colloidal osmotic pressures, COP) of the bHb-HES 130-containing solution obtained in example 1 and the bHb-HES 200-containing solution obtained in example 2 were each measured three times with a Wescor 4420 colloid osmometer at room temperature, the concentration of HES130 in the bHb +HES130 blend being equal to that in the bHb-HES 130-containing solution obtained in example 1, and the concentration of HES200 in the bHb +HES 200-containing solution obtained in example 2 being equal. The instrument was calibrated using Osmocoll (Wescor) standard solution and the results are shown in fig. 6.
FIG. 6 shows that COP values of bHb-HES130 and bHb-HES200 increase with concentration of bHb, and that the colloid osmotic pressures of bHb-HES130 and bHb-HES200 are both significantly higher than those of bHb, a blend of bHb+HES130, and a blend of bHb +HES 200. It can be seen that both conjugates can expand and maintain intravascular blood volume, thereby improving blood circulation in hypoxic tissue.
Experiment seven, erythrocyte stiffness index
The deformability of the red blood cells reflects the ability of the red blood cells to improve microcirculation through capillaries, and the good denaturation ability of the red blood cells is beneficial to improving microcirculation through capillaries, whereas the poor denaturation ability is not beneficial to improving microcirculation through capillaries. The erythrocyte stiffness index (Index of rigidity, IR) is an index for evaluating deformability of erythrocytes, and a large erythrocyte stiffness index indicates poor deformability of erythrocytes. The test thus evaluates the effect of bHb-HES130 and bHb-HES200 on the ability of erythrocytes to deform by measuring the erythrocyte stiffness index.
The method comprises the following steps: the whole blood of rat was mixed with physiological saline (NS), PB solution of bHb, bHb-HES 130-containing solution obtained in example 1 and the like in a volume ratio of 5:1, respectivelyThe bHb-HES 200-containing solution obtained in example 2 was mixed as a whole blood sample, and the total blood sample was 14.8g/100mL in terms of the final concentration of hemoglobin; rat plasma was mixed with physiological saline (NS), bHb, the bHb-HES 130-containing solution obtained in example 1 and the bHb-HES 200-containing solution obtained in example 2, respectively, at a volume ratio of 5:1, as plasma samples. The viscosity of each sample was then measured using a us boler viscometer (model: DV 3T) while the viscosity of whole blood and plasma was measured (results see table 2), substituting the viscosity results for each sample into the formula ir= (η) h -η p )/η p The rigidity index IR was obtained by X (1/Hct) wherein Hct represents the hematocrit (the hematocrit of whole blood was 41.6% and the hematocrit of whole blood sample was 34.9%), η h And eta p At high shear rates (200S for whole blood samples and plasma samples, respectively -1 ) The following viscosity and the results are shown in Table 2 and FIG. 7.
TABLE 2 viscosity of the samples of each group at different shear rates
The results in Table 2 show that the viscosity of each whole blood sample group was lower than that of whole blood (blood group), indicating that the addition of physiological saline, bHb-HES130, bHb-HES200 did not increase the blood viscosity and did not affect the normal flow of blood. Compared with normal saline and bHb, bHb-HES130 and bHb-HES200 can reduce blood viscosity more, which indicates that the conjugate of the invention has the function of improving interstitial blood fluidity and promoting microcirculation.
FIG. 7 shows that the erythrocyte stiffness indices of the blood, NS, bHb, bHb-HES130, and bHb-HES200 groups are 12.25, 10.41, 10.44, 6.85, and 6.20, respectively. It can be seen that bHb-HES130 and bHb-HES200 can reduce rigidity index of erythrocytes and have remarkable effect after being mixed with blood, which indicates that bHb-HES130 and bHb-HES200 can remarkably improve deformability of erythrocytes after being infused into a body, and is beneficial to improving microcirculation of erythrocytes through capillaries.
Experiment eight: haemocompatibility-haemolysis experiments
Taking physiological saline, 30 mu L of PB solution of bHb, solution containing HES130, solution containing HES200, solution containing bHb-HES130 obtained in example 1 and solution containing bHb-HES200 obtained in example 2 respectively, mixing with red blood cells (300 mu L) respectively, so that the packed volume of red blood cells (Hct) is 30%, and incubating for 1h at 37 ℃ to obtain red blood cell suspension; 150. Mu.L of each of the red blood cell suspensions was mixed with 400. Mu.L of physiological saline, and the total hemoglobin concentration (ctHb) in the mixed system was measured by using a BC-500 veterinary fully automatic hemocytometer (Mindary, china). Centrifuging (2000 g,10 min) the mixed system, mixing the supernatant with a color reagent (purchased from Nanjing institute of biological engineering) in a free hemoglobin detection kit, incubating at 37 ℃ for 20 minutes to obtain a sample, measuring the absorbance of the sample at 510nm (using distilled water as a control), obtaining the concentration of free Hb, and repeating the measurement three times. The hemolysis ratio was calculated as formula hemolysis ratio=free Hb concentration× (1-Hct)/cthb×100%, and the results are shown in table 3.
TABLE 3 hemolysis ratio of bHb-HES130 and bHb-HES200
| Sample of | Rate of hemolysis/% | Sample of | Rate of hemolysis/% |
| Physiological saline | 0.039±0.001 | bHb-HES130 | 0.030±0.001 |
| bHb | 0.046±0.001 | bHb-HES200 | 0.042±0.001 |
The results in Table 3 show that the hemolysis rates of bHb-HES130 and bHb-HES200 are close to those of bHb and NS, indicating good blood compatibility of bHb-HES130 and bHb-HES200.
When the blood source is insufficient, special blood types exist, and the traffic is inconvenient in disaster, an air drop blood bag is needed, and the air drop with a certain height can cause hemolysis of blood to influence the infusion effect. The experiment shows that the bHb-HES130 and bHb-HES200 provided by the invention have good compatibility with blood, and can be used for air drop in emergency.
Experiment nine: blood compatibility-coagulation test
15. Mu.L of physiological saline, bHb PB solution, bHb-HES130 solution obtained in example 1 and bHb-HES200 solution obtained in example 2 (bHb concentration: 10. Mu.g bHb/. Mu.L) were mixed with platelet rich plasma PRP (210. Mu.L) and platelet poor plasma PPP (235. Mu.L), respectively, and the mixture was continuously stirred at 100rpm for 15 minutes at room temperature. All the mixtures were placed in a thermostated well of a platelet aggregation instrument (Helena AggRAM, usa), incubated at 37 ℃ for 15 minutes, and the instrument was calibrated with distilled water. The PPP-containing mixture was measured directly, and then 25. Mu.L of 50. Mu. Mol/L Adenosine Diphosphate (ADP) was added to the PRP-containing mixture, and the spectrum of the percentage of aggregation was recorded by using HemoRam software (version 1.3), and the maximum percentage of aggregation was directly obtained from the spectrum and reflected as the aggregation rate of platelets, and the results are shown in Table 4.
TABLE 4 bHb-HES130 and bHb-HES200 platelet aggregation levels
| Sample of | Platelet aggregation Rate/% | Sample of | Platelet aggregation Rate/% |
| Physiological saline | 78.3±4.8 | bHb | 80.4±0.5 |
| HES130 | 84.7±0.7 | bHb-HES130 | 92.8±1.8 |
| HES200 | 80.6±2.5 | bHb-HES200 | 91.7±1.4 |
Table 4 shows that the platelet aggregation levels of group bHb (80.4%), HES-130 (84.7%) and HES-200 (80.6%) were all similar to those of the NS group (80.7%). The blood platelet aggregation degree of the bHb-HES130 group (92.8%) and the bHb-HES200 group (91.7%) are higher than that of the NS group (80.7%) and the difference is obvious, which shows that the bHb-HES130 and the bHb-HES200 can promote blood platelet aggregation, and the bHb can not influence the blood coagulation function of the blood platelets after being coupled with the HES, can promote the blood platelets to play the blood coagulation function, and is beneficial to controlling the blood loss of hemorrhagic shock.
The foregoing is merely a preferred embodiment of the invention, and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended by the present invention.
Claims (14)
1. Application of hydroxyethyl starch hemoglobin conjugate in preparing plasma dilatant and treating hemorrhagic shock or severe anemia;
the hydroxyethyl starch hemoglobin conjugate is obtained by coupling hydroxyethyl starch with hemoglobin, the molecular weight of the conjugate is more than 200kDa, and the preparation steps of the conjugate comprise oxidizing hydroxyethyl starch (HES) with sodium periodate and coupling hemoglobin (Hb) with oxidized hydroxyethyl starch;
the molecular weight of the hydroxyethyl starch is 130-200kDa;
hb, oxidized HES and NaCNBH upon coupling 3 Mix, and Hb: oxidized HES: naCNBH 3 The molar ratio of (2) is 4:3:300;
the plasma expander or the medicine can be applied to an acute blood transfusion link to partially or completely replace plasma to play a role in increasing blood volume, and simultaneously partially or completely replace red blood cells to play a role in carrying oxygen and releasing oxygen, and the oxygen partial pressure P when the oxygen saturation is 50 percent 50 The erythrocyte rigidity index is 5-6 at 10-20mmHg, and the colloid osmotic pressure is 5-30mmHg when the hemoglobin concentration is 5-25 mg/mL.
2. The use according to claim 1, wherein the hemorrhagic shock is shock caused by trauma, peptic ulcer, esophageal varices vein rupture, gynaecological and obstetrical diseases, perioperative induced blood loss.
3. The use according to claim 1, wherein the acute transfusion session is an acute transfusion session of blood loss, apheresis, stabilization of blood circulation and dilution of blood.
4. The use according to claim 1, wherein the erythrocyte stiffness index is 5.10-5.80 after mixing the medicament with blood.
5. The use according to claim 1, wherein the plasma expander has a gel osmotic pressure of 7.5-27.5mmHg at a hemoglobin concentration of 5-25 mg/mL.
6. The use of claim 1, wherein the hemoglobin in the conjugate is in a tetrameric structure.
7. The use of claim 1, wherein the hemoglobin secondary structure of the conjugate is intact.
8. The use according to claim 1, wherein the conjugate has characteristic peaks at the 260nm, 285nm and Soret bands in a circular dichroism spectrum.
9. The use according to claim 1, wherein the conjugate has three characteristic peaks at 410nm, 540nm and 576nm in the uv-vis spectrum.
10. The use of claim 1, wherein the conjugate has no peak at 630nm in the uv-vis spectrum.
11. The use according to claim 1, wherein the conjugate is 3300cm in an infrared spectrum -1 、2925cm -1 、2851cm -1 、1650cm -1 And 1540cm -1 There is a characteristic peak.
12. The use of claim 1, wherein the conjugate is at 1540cm -1 Point and 1650cm -1 The characteristic peak intensity at the site is stronger than that of hemoglobin.
13. Use according to claim 1, characterized in that the haemoglobin is selected from human haemoglobin, bovine haemoglobin or porcine haemoglobin.
14. The use according to claim 1, wherein the conjugate is bHb-HES130 and/or bHb-HES200.
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