JPH03291239A - Hemoglobin-containing liposome - Google Patents

Abstract

PURPOSE:To obtain the title liposome useful irrespective of various infectious diseases and blood types, having excellent oxygen transporting ability and safety, by putting an aqueous solution of hemoglobin containing a phosphoric acid condensate in liposome having a membrane composed of a lipid. CONSTITUTION:An aqueous solution of hemoglobin containing a phosphoric acid condensate (preferably metaphosphoric acid condensate or polyphosphoric acid condensate having 2-6 degree of condensation), an allosteric factor to gradually decompose in body into a safe substance is stored in liposome composed of a lipid, preferably phospholipid to give artificial erythrocyte. Hemoglobin is taken in a state of an aqueous solution and the concentration thereof is 30-60wt.%. The phosphoric acid condensate used is 10mug to 10mg based on 1mg hemoglobin. The phosphoric acid condensate is added to a membrane comprising lipid constituting liposome and further hemoglobin chemically modified with polyphosphoric acid may be used.

Description

[Detailed description of the invention]

[0001]

[Industrial application field]

The present invention relates to hemoglobin-containing liposomes used, for example, as artificial red blood cells, and in particular to hemoglobin-containing liposomes that have excellent oxygen transportability and safety, and can be used as blood for transfusion without being affected by various infections or blood types. [0002]

[Conventional technology]

Conventionally, fluorocarbon, which has a physically high oxygen solubility, has been used as artificial red blood cells. Fluorocarbon is used in the form of a water emulsion, but in this case, the oxygen carrying capacity of the fluorocarbon emulsion is only 1/3 that of blood. Therefore, during use, it is necessary to perform respiratory control using an oxygen tent, and there is also a problem in terms of toxicity, such as damage to the kidneys and the like. [0003] On the other hand, the human allosteric factor is 2,3-diphosphoglycerate (hereinafter abbreviated as 2,3-DPG), which is extremely unstable and remains in the blood taken from the body for several days. It can only exist for a short period of time. Therefore, most of the blood currently used for blood transfusions lacks oxygen-carrying ability, which poses a problem in blood transfusion therapy in cases of heavy bleeding. Therefore, various artificial red blood cells that can enhance oxygen carrying capacity have been proposed. JP-A No. 57-26621 discloses a structure in which inosit hexaphosphate derivatives, nucleotide phosphate derivatives, polycarboxylic acids, hexacyanoferrates, etc. are added as allosteric factors to artificial red blood cells to enhance the oxygen carrying capacity of the blood. Disclosed. Also, JP-A-64-6142
No. 6 discloses a structure in which the above-mentioned allosteric factor is made into a liposome together with hemoglobin. Note that, from the viewpoint of oxygen transport ability, inosit hexaphosphoric acid has attracted the most attention among allosteric factors in the above-mentioned prior art. [0004] On the other hand, JP-A-61-37735 proposes a structure in which a methemoglobinization inhibitor is incorporated in addition to hemoglobin into a liposome. Here, the inclusion of a methemoglobinization inhibitor prevents a decrease in oxygen transport ability due to oxidation of hemoglobin to methemoglobin. Similarly, JP-A-62-178521
In this issue, the conversion of hemoglobin to methemoglobin is prevented by incorporating vitamin C into an aqueous hemoglobin solution. [0005] On the other hand, JP-A-63-66117 discloses a method for producing liposomes incorporating a highly viscous aqueous solution, in which a hemoglobin aqueous solution having a hemoglobin concentration close to that of natural red blood cells is incorporated into liposomes. It has been fulfilled,
This increases oxygen carrying capacity. [0006]

[Problem to be solved by the invention]

However, in the conventional methods described above, except those using allosteric factors, the oxygen carrying capacity is still insufficient, and it has been difficult to put them into practical use as artificial red blood cells. Furthermore, inosit hexalinic acid, which has attracted attention as an allosteric factor, is originally an allosteric factor in birds. Therefore, inodite hexaphosphoric acid has 2.3
-Although it is much more stable than DPG, it is considerably more toxic because it is not originally present in human blood. Therefore, problems remain in terms of safety when using artificial red blood cells containing Inojit hexaphosphate, particularly when it is necessary to administer large amounts of artificial blood in liter units, such as in blood transfusions. [0007] An object of the present invention is to provide a hemoglobin-containing material with low toxicity, which has an even higher oxygen carrying capacity, is suitable for use as an artificial red blood cell, and is particularly suitable for administration in large quantities as blood for transfusion. Our goal is to provide liposomes. [0008]

[Means to solve the problem]

In order to obtain artificial red blood cells that are suitable for administration in large quantities as in the case of blood transfusion therapy, the inventors of the present application have intensively investigated allosteric factors that gradually decompose in the body and become safe substances. As a result, condensed phosphoric acid has a high oxygen-carrying ability and gradually decomposes into phosphoric acid in the body, so if this condensed phosphoric acid is used, it is possible to create artificial red blood cells with high safety and high oxygen-carrying ability. The present inventors have discovered that the present invention can be configured. That is, the present invention is a hemoglobin-containing liposome characterized in that an aqueous hemoglobin solution is incorporated into a liposome having a membrane made of lipid, and a phosphoric acid condensate is contained in the aqueous hemoglobin solution. Note that a liposome is defined as a closed vesicle having a membrane mainly composed of lipids. [0009] The liposome membrane-forming lipid used in the present invention is not particularly limited, and any type of natural or synthetic lipid can be used as long as it can form liposomes. In particular, phospholipids are preferably used, and examples thereof include lecithin phosphadylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, cardiovirin, and those obtained by hydrogenating these in a conventional manner. Moreover, it is also possible to use the various phospholipids exemplified in appropriate combinations. [0010] The liposomes used in the present invention are produced by suspending a lipid solution and then ejecting it under high pressure. The step of high-pressure discharge treatment of the raw material solution is, for example, using a French press to process the raw material solution at a rate of 50 to 2500 kg/Cm2, preferably 500 kg/Cm2.
This is done by discharging several times through a narrow gap under a pressure of ~1500 kg/Cm2. The higher the pressure, the smaller the particle size of the resulting liposomes. [0011] Hemoglobin to be incorporated into liposomes is prepared by treating red blood cells in a conventional manner. for example,
Red blood cells are hemolyzed and concentrated to 20% (W/W) or more by ultrafiltration using a membrane with a molecular weight cutoff of 50,000. Further, hemoglobin is incorporated in the form of an aqueous solution, and its concentration is 30 to 60% (W/W). [0012] The phosphoric acid condensate used in the present invention includes (HBO
2) Metaphosphoric acid represented by n (n is an integer of 2 or more), 2
Examples thereof include polyphosphoric acids containing atomic or more phosphorous and having P-0-P bonds, and analogs thereof. Examples of the metaphosphoric acid include trimetaphosphoric acid (I) and tetrametaphosphoric acid (II), and examples of the polyphosphoric acid include pyrophosphoric acid (III) and tetrapolyphosphoric acid (IV). [0013]

[Chemical formula 1] (I) ■ H [0014] 0H [0015]

[Chemical formula 3] (III) [0016]

embedded image (IV) [0017] Analogs related to the above metaphosphoric acid and polyphosphoric acid include the above compound (I). Specifically, the following compounds (
V) (VII) is exemplified. [0018]

[C5] (V) [0019] 0 = P - 0) I Fortune H [0020]

embedded image (VIl) [00213 In addition to the above-mentioned compounds (I) to (VII), compounds that produce phosphoric acid condensates when dissolved in water, such as phosphorus pentoxide, can also be used in the present invention. Can be done. When phosphorus pentoxide is dissolved in water, it undergoes hydrolysis to form tetraultrapolyphosphoric acid (VII) and tetrametaphosphoric acid (I).
I), tetrapolyphosphoric acid (IV), etc. However, the final product of hydrolysis, orthophosphoric acid (H3P0
In case 4), the effect of the present invention is not recognized. [0022] The degree of condensation of the phosphoric acid condensate is 2 to 2 from the viewpoint of oxygen carrying capacity.
Most preferably, it is in the range of 6. However, even compounds with a higher degree of polymerization can be used in the present invention because they produce phosphoric acid condensates of P2 to P6 by hydrolysis in an aqueous solution. The above-mentioned phosphoric acid condensate can obtain the same effect even if it is a salt thereof. The type of salt is not particularly limited. For example, salts of alkali metals and alkaline earth metals can be exemplified, and Na salts, Na salts, etc. are preferably used. [0023] The usage amount of the phosphoric acid condensate and its salts is hemoglobin 1
The ratio is 10 μg to 10 mg. (For example, stearic acid) can be added to strengthen the membrane structure or adjust the disintegration time. [0024] Further, in the present invention, the above-mentioned phosphoric acid condensate and/or its salt may be contained in the lipid membrane constituting the liposome, and furthermore, as hemoglobin incorporated into the liposome, Hemoglobin chemically modified with polyphosphoric acid may also be used. In that case, the oxygen carrying capacity of hemoglobin is effectively enhanced by the action of the phosphoric acid condensate on hemoglobin, as described above. [0025] When using a method of containing a phosphoric acid condensate in the lipid membrane or a method of chemically modifying hemoglobin, the amount of the phosphoric acid condensate used includes the amount contained in the hemoglobin aqueous solution. In the liposome, the total amount of phosphoric acid condensate is 10 μg to 10 mg per 1 mg of hemoglobin. Although not included in the scope of the present invention, a method of containing a phosphoric acid condensate in a lipid membrane without containing a phosphoric acid condensate in an aqueous hemoglobin solution, and a method of chemically modifying hemoglobin with polyphosphoric acid are also available. Even when used alone, the oxygen carrying capacity can be increased in the same way as in the present invention. The effect of improving the oxygen carrying capacity of hemoglobin by the above-mentioned salt-containing phosphoric acid condensate or phosphorus forge condensate salt is thought to be due to the nature of the 2,3-DPG pocket on hemoglobin. 2.3- Regarding the DPG pocket, Bene
(Biochem, Biophys, Res
, Commun, 26, 162.1967):C
hautin et al. (Arch, Biochem, Biop
hys, Eng. A1:96, 1967) and others. [0026] 2.3-DPG pocket is formed by basic amino acid residues such as histidine and lysine of hemoglobin β chain, and N-terminal valine of hemoglobin β chain of hemoglobin Ale, and has cationic properties. It is known. The phosphoric acid condensate and its salts used in the present invention have anionic properties and appropriate molecular shapes, and thus have a strong affinity for the 2.3-DPG pocket. Therefore, it is thought that the oxygen carrying capacity of hemoglobin is enhanced by the allosteric effect. [0027]

【Effect of the invention】

According to the present invention, since the phosphoric acid condensate is contained in the hemoglobin aqueous solution incorporated into the liposome, the oxygen carrying ability of hemoglobin is effectively enhanced. Furthermore, in a toxicity test, the phosphoric acid condensate has a toxicity of about 115 at a 50% lethal dose compared to inosit hexaphosphoric acid, and can be said to be a safer allosteric factor. Therefore, it is possible to realize artificial red blood cells that are safer and have a higher oxygen carrying capacity. The hemoglobin-containing liposome of the present invention is used as an artificial red blood cell in the same manner as conventionally known hemoglobin-containing liposomes. That is, the liposomes of the present invention can be stored by lyophilization and dispersed in physiological saline or plasma substitute at the time of use. Liposomes dispersed in plasma have a high oxygen carrying capacity and can be effectively used as artificial red blood cells. In particular, since the phosphoric acid condensate has low toxicity, the artificial blood using the hemoglobin-containing liposome of the present invention is suitably used when large-scale blood transfusion is required. [0028]

【Example】

Hereinafter, non-limiting examples of the present invention will be described. 100cm of blood was collected from a healthy person's vein using a blood collection tube containing an anticoagulant.
Collect whole blood from c. The obtained whole blood was centrifuged at 3,000 rpm for 30 minutes to remove the upper layer of plasma, platelets, and white blood cells, and the red blood cell sediment layer was resuspended using physiological saline.
Perform centrifugation at rpm for 30 minutes. [0029] Obtain 20 ml. Washed red blood cells obtained from five healthy individuals in the same manner as above are mixed to obtain a total of 100 ml of washed red blood cells. Next, twice the volume of washed red blood cells, ie, 200 ml of distilled water is added to hemolyze the red blood cells. Next, after adjusting the pH to 5.85 with IN HCI solution,
Centrifuge at 7000 rpm for 30 minutes. The supernatant was filtered through a membrane filter with a pore size of 0.22 μm,
Obtain 240 cc of hemoglobin liquid from which red blood cell membranes have been removed. Hemoglobin concentration was 5.5% (W/W). [00303 The obtained hemoglobin solution was ultrafiltered using a membrane with a molecular weight cutoff of 50,000 to a hemoglobin concentration of 30% (
W/W) to obtain 40 ml of red blood cell membrane-removed hemoglobin at a concentration of 30% (W/W). Liposome/month 11 1.475 g of purified phosphatidylcholine, 0.928 g of cholesterol, and 0.098 g of stearic acid are dissolved in chloroform. The obtained lipid solution is put into an eggplant flask, and evaporation is performed to remove chloroform and form a lipid film on the bottom of the eggplant flask. 40 ml of red blood cell membrane-removed hemoglobin with a hemoglobin concentration of 30% (W/W) to which 0.06 g of tetrapolyphosphoric acid (manufactured by Taihei Kagaku Co., Ltd.) was added was added to the lipid membrane obtained as described above, and shaken or Make a homogeneous suspension using sound waves. This raw material solution was heated to 700k using a French press.
Treat three times at a pressure of g/Cm2. The raw material solution after French press treatment was added to 40ml of physiological saline.
After diluting 5 times with physiological saline to which 0.06 g of tetrapolyphosphoric acid has been added, ultracentrifugation is performed at 50,000 rpm for 90 minutes to remove excess hemoglobin and excess lipids in the upper layer. [0031] With the physiological saline added with the above tetrapolyphosphoric acid,
Suspend the hemoglobin-containing liposome precipitate and
Perform ultracentrifugation at 0000 rpm for 90 minutes. The hemoglobin-containing liposomes were finally resuspended in saline (artificial red blood cell value). Liver value (I) Allosteric effect of each allosteric factor and phosphoric acid condensate The hemoglobin solution from which the red blood cell membrane was removed was added to pH 6, 0, 0, 1 mM B15-Tris buffer to make a 15.5 μM hemoglobin solution. Prepared. The obtained hemoglobin solution was mixed with 62 μM of allosteric factors (allosteric factors were not added in the case of control), and the oxygen dissociation curve of each allosteric factor was measured at 25° C. using a Hemotax Analyzer (manufactured by TC3, USA). The results are shown in Figure 1. [0032] In FIG. 1, the symbols marked on each curve indicate that the following are used as allosteric factors. ...Control, ×...2,3-DPG,...
・Tripolyphosphoric acid, mouth...tetrapolyphosphoric acid, ■...
- Hexametaphosphoric acid, ○... Inosit hexaphosphoric acid. The oxygen dissociation curve represents the oxygen carrying capacity of hemoglobin, and the more the curve is shifted to the right, the higher the oxygen carrying capacity. As is clear from Figure 1, all condensed phosphoric acids have a higher oxygen carrying capacity than the control, tripolyphosphoric acid has the same ability as 2,3-DPG, which is a human allosteric factor, and tetraphosphoric acid and hexametaphosphoric acid have a higher oxygen carrying capacity than the control. Dithexalinic acid (
Although it is slightly inferior to IHP), it is still very high. Further, Table 1 below shows that orthophosphoric acid, which has a 50% higher acidity than various polyphosphoric acids and has a degree of condensation n of 1, does not have this ability. [0033]

[Table 1] [0034] (II) Toxicity test of polyphosphoric acid and IHP Using ddY mouse (SPF) male, 7 weeks old, tetrapolyphosphoric acid and IHP were tested.
Dissolve each HP in sterile physiological saline and administer 10 mg to 500 mg per 1 kg of mouse body weight from the tail vein of the mouse.
As a result of administration within the range of It was about 175. (III) Oxygen dissociation property of liposomal hemoglobin The oxygen dissociation curve of the hemoglobin-containing liposome of Example containing tetrapolyphosphoric acid obtained as described above was measured using a dissolved oxygen measuring device 5TAT profile 5
(manufactured by Nippon Technicon). For comparison, a hemoglobin-containing liposome containing no tetrapolyphosphoric acid was prepared as a comparative example, and an oxygen dissociation curve was similarly determined. The comparative example was produced in the same manner as described above, except that 0.06 g of tetrapolyphosphoric acid was not added. [0035] Oxygen dissociation curves of Examples and Comparative Examples are shown in FIG. As is clear from FIG. 2, it can be seen that the oxygen dissociation curve of the hemoglobin-containing liposome containing tetrapolyphosphoric acid (Example) is shifted to the right compared to the hemoglobin-containing liposome of the comparative example. 100mmHg and 40m
Focusing on the oxygen bonding rate at mHg, in the comparative example,
They are ioo% at 100 mmHg and 65% at 40 mmHg, respectively, whereas in the example, they are 100% at 100 mmHg and 40% at 40 mmHg. [0036] The partial pressure of oxygen in the human alveoli is 100 mmHg, and in the peripheral system it is about 40 mmHg, so in the comparative example, the oxygen partial pressure was 35%
In the example, 60% is the amount of oxygen transported. Therefore, it was confirmed that the hemoglobin-containing liposomes of the above examples can dramatically increase the oxygen carrying capacity compared to the comparative examples.

[Brief explanation of drawings]

[Figure 1] FIG. 3 is a diagram showing oxygen dissociation curves of each allosteric factor.

FIG. 2 is a diagram showing oxygen dissociation curves for hemoglobin-containing liposomes of Examples and Comparative Examples.

【Document name】

drawing

[Figure 1]

[Figure 2]

Claims (1)

[Claims] Claim 1: In a liposome having a membrane made of lipid,
A hemoglobin-containing liposome comprising an aqueous hemoglobin solution and a phosphoric acid condensate contained in the hemoglobin aqueous solution.