阅读说明：本技术 交联血红蛋白的制备方法及包含该血红蛋白的器官灌注液 (preparation method of cross-linked hemoglobin and organ perfusate containing hemoglobin ) 是由 游可为 史国营 孙新宇 陈浩源 于 2019-09-09 设计创作，主要内容包括：本发明公开了一种交联血红蛋白的制备方法,其包括如下步骤：a、稀释脱氧血红蛋白,加入到无氧反应器中；b、无氧反应器顶部设置雾化器,交联剂通过所述雾化器加入反应器中,发生交联反应；c、加入还原剂终止交联反应,用超滤换液的方式除去未反应的交联剂和终止还原剂,得到交联血红蛋白。本发明制得的交联血红蛋白含有较低含量的大分子(大于500kD)和小分子(小于64KD),加入到器官灌注液中能够解决现行灌注液因缺少携氧剂而引起器官的酸中毒和溶酶体酶激活不良后果及再灌注时产生过量的自由基损伤细胞的现象,其制备、储存、运输及使用均十分简单方便。(The invention discloses a preparation method of cross-linked hemoglobin, which comprises the following steps: a. diluting the deoxyhemoglobin, and adding the diluted deoxyhemoglobin into an anaerobic reactor; b. an atomizer is arranged at the top of the anaerobic reactor, and a cross-linking agent is added into the reactor through the atomizer to perform a cross-linking reaction; c. adding a reducing agent to terminate the crosslinking reaction, and removing unreacted crosslinking agent and terminating the reducing agent by an ultrafiltration liquid exchange mode to obtain the crosslinked hemoglobin. The cross-linked hemoglobin prepared by the invention contains low-content macromolecules (more than 500kD) and small molecules (less than 64kD), can solve the problems of organ acidosis and lysosome enzyme activation badness caused by lack of oxygen carrying agent of the existing perfusate and the phenomenon of cell damage caused by excessive free radicals generated during reperfusion after being added into the organ perfusate, and is very simple and convenient in preparation, storage, transportation and use.)

1. A method for preparing cross-linked hemoglobin, comprising the steps of:

a. Diluting the deoxygenated hemoglobin, and adding the diluted deoxygenated hemoglobin into an anaerobic reactor;

b. An atomizer is arranged at the top of the anaerobic reactor, and a cross-linking agent is added into the reactor through the atomizer to perform a cross-linking reaction;

c. Adding a reducing agent to terminate the crosslinking reaction, and removing unreacted crosslinking agent and terminating the reducing agent by an ultrafiltration liquid exchange mode to obtain the crosslinked hemoglobin.

2. the method for preparing cross-linked hemoglobin according to claim 1, wherein in step a, the concentration of deoxyhemoglobin is diluted to 1-3g/dl, and a mercapto compound is added to the hemoglobin so that the mass ratio of hemoglobin to mercapto compound in the solution is 5-10: 1.

3. The method of claim 2, wherein in the step a, the sulfhydryl compound is mercaptoethylamine, glutathione or N-acetyl-L-cysteine.

4. The method of claim 1 or 2, wherein the crosslinking agent is added at a rate of 50 to 500ml/min in the step b.

5. The method of claim 1, wherein in step b, the pressure in the reactor is 1.05-1.25 bar.

6. The method of claim 1, wherein the mass ratio of hemoglobin to cross-linking agent added to the reactor is 1000: 29-45.

7. an organ perfusate comprising the cross-linked hemoglobin prepared by the method of any one of claims 1 to 6.

8. The organ perfusate of claim 7, wherein the organ perfusate further comprises vitamin K1 and prostacyclin.

9. the organ perfusate of claim 8, wherein the organ perfusate comprises:

The contents of the above components refer to the contents in 1000ml of the organ perfusate.

10. a method of preparing the organ perfusate of claim 9, comprising the steps of:

a. adding human albumin, heparin, 8.4% of sodium bicarbonate, 10% of calcium gluconate, vancomycin, gentamicin, 10% of compound amino acid injection, 12 compound vitamins for injection and vitamin K1 according to the formula proportion, quantifying distilled water, filtering for sterilization, aseptically filling and sealing to obtain a solution I;

b. Subjecting the cross-linked hemoglobin solution prepared by the method of any one of claims 1-6 to deoxygenation, filtration sterilization, aseptic and anaerobic filling, and thermoplastic sealing to obtain solution II;

c. providing separately packaged prostacyclin according to a formula proportion to obtain a solution III;

d. and uniformly mixing the solution I and the solution II according to a ratio, and performing infusion by matching with the solution III to obtain the organ perfusate.

Technical Field

the invention relates to the technical field of organ preservation in medical technology, in particular to a preparation method of cross-linked hemoglobin, and especially relates to an organ perfusate containing the cross-linked hemoglobin.

Background

Many human diseases are caused by necrosis of cells, tissues and organs, thereby causing functional loss and causing disability and even death of human bodies, and with the development of modern medicine, organ transplantation technology is mature day by day, and the following diseases are caused only from the surgical view: the organ transplantation technology is perfect, and head replacement surgery is not a big problem. In fact, organ transplantation still faces various difficulties, and preservation of isolated organs is one of them. Organ transplantation requires the transplantation of a living organ, and therefore, it is a prerequisite for successful transplantation that the organ for transplantation maintains intact anatomy and viability from the time of excision from the donor until its principal blood vessel is connected to the recipient's blood vessel. However, once any organ loses blood supply, the cells die in a short period of time at normal temperature (35-37 ℃) without the necessary oxygen and nutrients. The tolerable time is very short, such as 10-15 minutes for heart and liver and 45-60 minutes for kidney, and if the time is exceeded, it is difficult to restore function after transplantation. In clinical practice, it is required that the ischemia time at normal temperature is as short as possible, preferably not longer than 3-5 minutes, and at most not longer than 7-8 minutes. It is not possible at all to complete the graft surgery in such a short time. Therefore, it is necessary to try to maintain the activity of the isolated organ for a long time.

in order to prolong the preservation time of isolated organs, scientists have studied various methods, wherein cryopreservation is a method which is commonly used at present, but the cryopreservation can effectively prolong the preservation time of organs, and the damage to cells can be obvious when the cryopreservation is carried out. When metabolism is inhibited, ATP supply is not available to Na pump, and intracellular proteins produce oncotic pressure, which leads to cell edema. Intracellular proteins and non-permeable anions produce an osmotic tension of about 110-140 mOsm/kg. When the Na pump activity is inhibited by low-temperature ischemia, the cell membrane potential is lowered. Na and Cl enter the cell due to concentration gradient, and in order to maintain water balance inside and outside the semipermeable membrane, intracellular water is accumulated to cause cell edema.

in addition, the preservation of isolated organs is prolonged by adopting a perfusion mode, and the currently commonly used perfusion solutions comprise Krebs-Henseleit solution, St Thomas II solution, UW solution, Celsior solution and the like, but the perfusion solutions lack oxygen carrying agents, so that the perfused organs are always in an anoxic state. If the in vitro perfusate can not effectively supply oxygen to the isolated organ, the tissues and cells can be subjected to anaerobic glycolysis increased due to oxygen deficiency, and the adverse effects of acidosis and lysosomal enzyme activation are caused. Ensuring oxygen supply, maintaining minimal energy metabolism and removing metabolic waste products is an urgent problem to be solved in organ in vitro preservation.

Meanwhile, organs preserved under anoxic conditions cause significant secondary damage during their transplant reperfusion, and the mechanism by which restoring blood flow aggravates the damage of preserved organs is not completely understood, generally thought to be related to the damaging effects of oxygen free radicals, and under normal conditions, hypoxanthine, a metabolite of ATP, is rapidly converted to purine by Xanthine Oxidase (XOD) and then to uric acid, a middle product. At the same time, hypoxia inactivates or depletes endogenous antioxidants such as superoxide dismutase (SOD), which can produce excessive free radicals to damage cells when the blood circulation reestablishes oxygen supply suddenly.

Therefore, it is important to add an oxygen-carrying agent to the organ perfusate. Erythrocytes are natural oxygen carriers in animals and have been used directly by humans for organ perfusion preservation in vitro. While erythrocytes are easily ruptured, extravasation of intramembrane aminophospholipids, endotoxins, glycophorins and intracellular enzymes can damage vascular endothelium and cardiomyocytes and cause capillary obstruction.

the adoption of stroma-free hemoglobin to replace red blood cells is a new idea. However, hemoglobin free of erythrocytes is nephrotoxic and can be clinically applied only by being crosslinked into protein with proper molecular weight, and crosslinked hemoglobin has methods such as PEG, glutaraldehyde, raffinose, aspirin and the like, wherein glutaraldehyde has the characteristics of high activity, quick reaction, high supply quantity, good crosslinking performance, stable property of a crosslinked product, capability of keeping a fine structure of protein and small influence on the activity of enzyme by virtue of the reaction of glutaraldehyde and protein, and becomes a method often selected for hemoglobin crosslinking. But glutaraldehyde crosslinking has obvious disadvantages, namely, the crosslinking reaction is not specific, because about 40 lysine side chain amino groups and four chain end amino groups on the surface of hemoglobin can participate in the reaction, the reaction is difficult to control, and the molecular weight distribution of the final product is from 32kD to over 1000kD, so that the final product is a non-uniform mixture. The control of molecular weight is always a difficult point of glutaraldehyde cross-linking hemoglobin, the too low molecular weight has poor stability, is easy to dissociate to cause nephrotoxicity, and has low oxygen carrying efficiency; too high a molecular weight increases the viscosity of blood, is not favorable for blood flow, and is deposited on the inner wall of blood vessels and in the liver or spleen. In vitro experiments show that products with high safety and effectiveness are obtained by reducing the content of unpolymerized dimer and reducing the content of macromolecular polymerization larger than 500kD as much as possible. Meanwhile, the cross-linked hemoglobin can replace red blood cells for organ perfusion preservation in vitro.

Disclosure of Invention

The invention aims to solve the technical problem of providing a preparation method of cross-linked hemoglobin, the cross-linked hemoglobin prepared by the method contains low-content macromolecules (more than 500kD) and small molecules (less than 64kD), the cross-linked hemoglobin is added into an organ perfusate, the problems of organ acidosis, poor activation of lysosomal enzyme and cell damage caused by excessive free radicals generated during reperfusion due to lack of an oxygen carrying agent in the existing perfusate can be solved, and the preparation, storage, transportation and use of the cross-linked hemoglobin are very simple and convenient.

in order to solve the technical problem, the invention provides a preparation method of cross-linked hemoglobin, which comprises the following steps:

a. Diluting the deoxygenated hemoglobin, and adding the diluted deoxygenated hemoglobin into an anaerobic reactor;

b. An atomizer is arranged at the top of the anaerobic reactor, and a cross-linking agent is added into the reactor through the atomizer to perform a cross-linking reaction;

c. Adding a reducing agent to terminate the crosslinking reaction, and removing unreacted crosslinking agent and terminating the reducing agent by an ultrafiltration liquid exchange mode to obtain the crosslinked hemoglobin. Wherein the reducing agent can be sodium borohydride, and the ultrafiltration is a tangential flow purification of the cross-linked hemoglobin using a membrane or hollow fiber column of 10Kd to 100 Kd.

In the step a, the concentration of the deoxyhemoglobin is diluted to 1-3g/dl, and a sulfhydryl compound is added into the hemoglobin, so that the mass ratio of the hemoglobin to the sulfhydryl compound in the solution is 5-10: 1.

In the preparation method of the cross-linked hemoglobin, in the step a, the sulfhydryl compound is mercaptoethylamine, glutathione or N-acetyl-L-cysteine.

the method for preparing cross-linked hemoglobin, wherein in the step b, the adding speed of the cross-linking agent is 50-500 ml/min.

the method for preparing the cross-linked hemoglobin, wherein in the step b, the pressure of the reactor is 1.05-1.25 bar.

The preparation method of the cross-linked hemoglobin is characterized in that the mass ratio of the hemoglobin to the cross-linking agent added into the reactor is 1000: 29-45.

The invention also provides organ perfusate which comprises the cross-linked hemoglobin prepared by the preparation method.

The organ perfusate further comprises vitamin K1 and prostacyclin.

The organ perfusate comprises:

the contents of the above components refer to the contents in 1000ml of the organ perfusate.

The invention also provides a preparation method of the organ perfusion fluid, which comprises the following steps:

a. Adding human albumin, heparin, 8.4% of sodium bicarbonate, 10% of calcium gluconate, vancomycin, gentamicin, 10% of compound amino acid injection, 12 compound vitamins for injection and vitamin K1 according to the formula proportion, quantifying distilled water, filtering for sterilization, aseptically filling and sealing to obtain a solution I;

b. subjecting the cross-linked hemoglobin solution prepared by the method of any one of claims 1-6 to deoxygenation, filtration sterilization, aseptic and anaerobic filling, and thermoplastic sealing to obtain solution II;

c. Providing separately packaged prostacyclin according to a formula proportion to obtain a solution III;

d. and uniformly mixing the solution I and the solution II according to a ratio, and performing infusion by matching with the solution III to obtain the organ perfusate.

the preparation method of the cross-linked hemoglobin has the following beneficial effects:

1. According to the preparation method of the cross-linked hemoglobin, the cross-linking agent is added by the atomizer, so that the cross-linking process is obviously improved, the content of macromolecules and micromolecules in the cross-linked hemoglobin is reduced, the content of micromolecules with the molecular weight of less than 64KD in the cross-linked hemoglobin is less than 5%, the content of macromolecules with the molecular weight of more than 500KD in the cross-linked hemoglobin is less than 10%, further molecular weight separation and purification are not needed for the cross-linked hemoglobin, the complexity of the process is reduced, the cost is saved, the yield is improved, the polymerization effect is improved, the cross-linked hemoglobin is easier to amplify, the adding speed of the cross-linking agent is obviously improved, the adding speed of the cross-linking agent can reach 500ml/min, the cross-linking time is greatly shortened, and the production cost;

2. According to the preparation method of the cross-linked hemoglobin, the sulfhydryl compound is added before cross-linking, so that the contents of high molecular weight hemoglobin and low molecular weight hemoglobin are further reduced, the risks of nephrotoxicity and vasoconstriction caused by the low molecular weight hemoglobin and the increase of blood viscosity caused by the high molecular weight hemoglobin are reduced, the flow of blood is not facilitated, and the risks of deposition on the inner wall of a blood vessel and the liver or spleen are reduced;

3. according to the preparation method of the cross-linked hemoglobin, the content of macromolecules and micromolecules in the cross-linked hemoglobin is further reduced by improving the pressure of the reactor;

4. The cross-linked hemoglobin prepared by the invention is added into organ perfusate, so that the problems of acidosis and poor activation of lysosomal enzyme caused by hypoxia of isolated organs and cell damage caused by excessive free radicals generated during perfusion are solved;

5. The coagulant vitamin K1 and the vasodilator prostacyclin are added into the organ perfusion liquid, so that the effectiveness and safety of cross-linked hemoglobin during perfusion are improved better;

6. The organ perfusate prepared by the invention well solves the problem that hemoglobin is oxidized due to long-term storage by filling the perfusate under the deoxidation and anaerobic conditions and filling the perfusate by adopting a medicine bag with low oxygen permeability;

7. After the organ perfusate prepared by the invention is stored for 1 year, the activity detection is carried out on the contained cross-linked hemoglobin, and the content of oxygenated hemoglobin and the content of methemoglobin are both not higher than 5 percent and are both in a qualified range.

drawings

FIG. 1 is a schematic diagram of a process for preparing cross-linked hemoglobin according to the present invention.

Detailed Description

The present invention is described in detail below with reference to the drawings and examples.

preparation of crosslinked hemoglobin