JPH02121932A - Hemoglobin-containing liposome, preparation thereof and raw solution for same preparation - Google Patents

Abstract

PURPOSE:To provide a liposome capsuled with a single-layered two molecular membrane, obtained from a lipid comprising mainly a phospholipid and hemoglobin in predetermined amounts, respectively, having a thin membrane thickness and an uniform particle size and capable of being utilized as an artificial erythrocyte having a high oxygen-carrying ability and high safety. CONSTITUTION:A lipid comprising mainly a phospholipid in an amount W(mol/l) obtained by a calculation equation of formula I [r is the radius (Angstrom ) of a capsule; d is the membrane thickness (Angstrom ) of the capsule; N is the concentration (capsule/l) of the capsule; NA is Avogadro number; S is the average value (Angstrom <2>) of the molecular occupying area of the phospholipid] is homogeneously dispersed in and mixed with hemoglobin in an amount determined by a calculation equation of formula II {[Hb]i is the concentration (g/l) of Hb (hemoglobin) charge on the preparation of the capsule; [Hb]f is the final concentration of the Hb} to give an initial capsule, which is subjected to processes comprising a capsulation process by an extrusion method, a process for improving the packaging efficiency of the capsule by a freezing melting method and a process for controlling the size of the capsule by an extrusion method to provide a hemoglobin-containing liposome capsuled with a single layered two molecular membrane.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention relates to a hemoglobin-containing liposome, a method for producing the same, and a stock solution for the production method. The object of the present invention is to industrially provide hemoglobin-containing liposomes having a uniform particle size.

The hemoglobin-containing liposome according to the present invention can be used as an artificial red blood cell with high oxygen transport function and high safety.

[Conventional technology] The technology for encapsulating biopolymers such as proteins is
It plays an extremely important role in various fields such as medicine, pharmacy, engineering, food science, and cell engineering. There are already many (
Although these technologies have been developed and are also used for intracorporeal administration, they all have unresolved technical problems. In recent years, by using lipids mainly composed of phospholipids as capsule construction materials, capsules that are safe and can be administered to living organisms have been made. One of them is hemoglobin-containing liposomes.

The conventional method that forms the core of the technology for producing hemoglobin-containing liposomes is called the so-called thin film method. That is, liposome-forming lipids are dissolved in a suitable organic solvent such as chloroform, the solvent is distilled off from the resulting solution to form a thin lipid film, and an aqueous solution of hemolyzed hemoglobin is added to this thin film, followed by vigorous stirring to form multilamellar liposomes. This is a method of forming and then subjecting it to ultrasonic treatment (U.S. Patent No. 4,133.874).
. During storage of hemoglobin-containing liposomes produced by this thin film method, the heme iron of the hemoglobin in the liposomes may gradually oxidize and transform into methemoglobin, which may lose its oxygen transport function. Furthermore, a liposome membrane in which the main component is a hydrogenated phospholipid with a hydrogenation rate of 50% or more has been proposed (Japanese Patent Publication No. 211230/1983).

In addition, in the process of producing hemoglobin-containing liposomes (1
) Initial encapsulation process by physical stimulation [Portex (
+Glass Peas) B, P, Gaber, P, Ya
ger, J, P, 5heridan, and
E, L, Chang FEBSLett153, 2
85 (1983)), (2) Encapsulation process by extrusion method (B, P, Gaber, an
d M, C, Farmer Prog, Cl1n,
Biol, Res, 165.179 (1984
)) and (3) particle size control step by extrusion method (M, J, Hope, M, B, Bally,
G, Webb and P, R, Cu11is.
Biochin, Biophys, Acmua 812. 55 (1985)] is known to be used.

[Problem to be solved by the invention]

In any case, the disadvantages of the conventional thin film method are that the produced liposomes are multilayered, the viscosity of the hemoglobin-containing liposomes is high, the blood circulation dynamics are poor, and furthermore, there are many lipids that form the liposomes. The point is that it becomes toxic. Therefore, research has been conducted to form liposomes with a single bilayer membrane, but an industrially usable technology has not yet been established.

The technical problem of the present invention is to industrially establish a technology for forming a liposome membrane into a monolayer bimolecular membrane of lipids mainly composed of phospholipids.
The purpose of the present invention is to provide a highly safe hemoglobin-containing liposome with good blood circulation dynamics and extremely low toxicity at a low cost.

[Means for Solving the Problems] and [Operations] That is, the present invention consists of the following three inventions.

(1) Calculate the amount required to form a monolayer bimolecular membrane of lipids whose main component is phospholipid using formula 1 [where W is t (mol/mol/ l), r is capsule radius (in), Δd is capsule membrane thickness (person), N is capsule concentration (pieces/1), N
A is Avogadro's number (6,02
)] Calculate the amount of hemoglobin that can be contained in the liposomes formed by the process necessary to form a monolayer bilayer membrane of lipids containing phospholipids as the main component. ) [However, (Hb) is added to the Hb (
Hemoglobin) concentration (g/l), [Hba F is the final Hb 1 degree (g/l), r is the capsule radius (person), N is the capsule concentration (units/l)], and is calculated using the above formula 1. It is manufactured using a stock solution for producing hemoglobin-containing liposomes that is initially encapsulated by physical stimulation by uniformly dispersing and mixing a certain amount of lipids whose main component is phospholipid into the hemoglobin obtained using the above calculation formula 2. A hemoglobin-containing liposome encapsulated in a monolayer bilayer membrane of lipids whose main component is phospholipid.

(2) Calculate the amount necessary to form a monolayer bimolecular membrane of lipids whose main component is phospholipid using formula 1 NA-3 [where W is the amount of lipids whose main component is phospholipid] (mol/1), r is capsule radius (λ), Δd is capsule membrane thickness (people), N is capsule concentration (pieces/l), NA
is Avogadro's number (6,02X1023), S is the average value of the molecular occupied area of lipids whose main component is phospholipid (input 2
)] Calculate the amount of hemoglobin that can be contained in a liposome formed in an amount necessary to form a monolayer bilayer membrane of lipids containing phospholipids as the main component using the formula 2% Formula % ) [However, (Hb)i is Hb (
(hemoglobin) concentration (g/l), [Hb)F is the final H
b, il1 degree (g/l), r is capsule radius (person), N
is determined by the capsule concentration (capsules/l)], and the amount of lipids mainly composed of phospholipids determined by the above calculation formula 1 is uniformly dispersed and mixed with the amount of hemoglobin determined by the above calculation formula 2, and the mixture is subjected to physical stimulation. The main ingredient is phospholipid, which is made by subjecting the stock solution for producing initially encapsulated hemoglobin-containing liposomes to an encapsulation process using an extrusion method, an encapsulation efficiency improvement process using a freeze-thaw method, and a particle size control process using an extrusion method. A method for producing a hemoglobin-containing liposome encapsulated in a monolayer bilayer membrane of lipids.

(3) Calculate the amount required to form a monolayer bimolecular membrane of lipids whose main component is phospholipid using formula 1 [where W is the amount of lipids whose main component is phospholipid (mol/ I), r is capsule radius (in), Δd is capsule membrane thickness (person), N is capsule concentration (pieces/l), NA
is Avogadro's number (6,02X1023), and S is the average molecular area occupied by lipids whose main component is phospholipid (2 people).
)] Calculate the amount of hemoglobin that can be contained in a liposome formed in an amount necessary to form a monolayer bilayer membrane of lipids containing phospholipids as the main component using the formula 2% Formula % ) (However, (Hb)i is Hb (
Hemoglobin) concentration (g/l), (Hb)r is the final H
b1 degree (g/l), r is the capsule radius (person), N is the capsule concentration (capsules/l)], and the amount of lipids mainly composed of phospholipids determined by the above calculation formula 1 is calculated using the above calculation. A stock solution for producing hemoglobin-containing liposomes that is homogeneously dispersed and mixed with the amount of hemoglobin determined by Formula 2 and subjected to initial encapsulation by physical stimulation.

Next, the structure of the present invention will be explained in more detail along with its operation as follows.

Lipids mainly composed of phospholipids used as capsules aggregate in water to form an ultra-thin film called a bilayer membrane. Depending on the conditions, they form a three-dimensionally closed hollow sphere (capsule), but the amount of lipids mainly composed of phospholipids used depends on the number of layers of lipids mainly composed of phospholipids (
n), capsule radius (r), capsule concentration (pieces/I) (
N). When determining the radius of a capsule, the most influential factor is the number of lipid membranes forming the capsule (number of layers). Connotation becomes possible. Furthermore, since hemoglobin in human blood is coated with a single-layer bimolecular membrane, it is ideal not only for efficient encapsulation but also physiologically. In this case, the amount of lipids whose main component is phospholipid can be calculated geometrically.

That is, if the physicochemical constants of lipids For example, the amount of lipids whose main component is the minimum phospholipid used is calculated from the molecular weight of the lipids whose main component is phospholipid and the above-mentioned constants, and becomes a function of n and N. It is sufficient to mix the thus determined amount of fat whose main component is phospholipid into the encapsulated hemoglobin (Hb) solution.

Since the amount of encapsulated hemoglobin solution is the amount that can be included in the capsule, this can also be calculated.

High homogenization of capsules is important, especially when they are intended for internal administration. For example, in the case of blood administration, uneven particle size may lead to thrombus formation in capillaries, resulting in a fatal defect. A stock solution for producing hemoglobin-containing liposomes is obtained by uniformly dispersing and mixing the lipids in the amount of hemoglobin determined by the above calculation formula 2, and is initially encapsulated by physical stimulation. In addition to the efficiency improvement step, the product is subjected to a particle size control step using an extrusion method, that is, a membrane filter is used to promote encapsulation with shear force, and the particle size of the produced capsules is selected. By this operation, capsules larger than the pore size of the filter used can be removed from the system. Naturally, capsules with a particle size smaller than the pore size will remain in the system, but
This can be solved by employing the freeze-thaw method described above. That is, capsules made of lipids mainly composed of phospholipids have the characteristic that the smaller the particle size, the more unstable they are and the easier they are to fuse. Therefore, by repeating the freeze-thaw operation, the particle size of the capsules increases and becomes uniform. Naturally, the capsules in the first part are larger than the set particle size, so they are filtered again to complete the particle size adjustment and added to the finished product.

A problem when encapsulating hemoglobin is that even if the starting hemoglobin solution is highly concentrated, it is diluted during the encapsulation stage. In order to avoid this, it is preferable to directly mix a high-concentration hemoglobin solution and lipids mainly composed of phospholipids, perform encapsulation in a high-concentration state, and complete the encapsulation. In order to increase the encapsulation efficiency, a freeze-thaw method is used to minimize the difference in hemoglobin concentration inside and outside the capsule.

The concentration of the contained hemoglobin solution can be adjusted freely.

The more concentrated the solution is, the more concentrated the hemoglobin solution will be. However, if it exceeds 50 wt%, the viscosity is high and handling is inconvenient.

Phospholipids include lecithin, soybean lecithin, corn lecithin,
Natural phospholipids such as cottonseed oil lecithin, rapeseed lecithin, etc.
Hydrogenated natural phospholipids are preferred, but the invention is not limited to natural phospholipids.

Many methods have been proposed for stabilizing capsules, among which polymerization of membranes through polymerization of components is effective. Of course, it is a requirement that there be no change in the molecular packing state of the components before and after polymerization, but this is due to the polymerizable phospholipid (D) used in the examples of the present invention.

It has already been physicochemically proven that there is no problem with DPC). Additionally, there is a concern that the polymerization process itself may cause denaturation of the encapsulated polymer, but this is also possible due to the activation of the less toxic radical polymerization initiator with ultraviolet light.
This can be avoided by obtaining a stable polymer capsule by using a low-temperature photoinitiated polymerization method or by crosslinking after encapsulation using polymerized phospholipids that have been selectively polymerized in advance.

It is known that lipids such as cholesterol are added to increase the strength of the capsule, and phosphatidic acid and the like are added to prevent capsule aggregation, but these can of course be adopted in the present invention.

〔Example〕

The present invention will be explained below by way of examples.

Example 1 Retention capacity 7.2 (1/mol) (L) at 130 nm
ipid (lipid)) r = 6.2 wt% (Hb) M = 15 wt% (Hb) F = 8.5 company% (r = 1300 people, Δd = 37 people, 5 = 58 people, N =
3.0 x 10" pieces/l) 30 g of egg yolk lecithin was mixed with 300 cc (15 wt%) of a physiological saline solution containing 45 g of hemoglobin, and treated with a homogenizer at 4°C for 3 hours under anoxic conditions to uniformly disperse the mixture. By mixing, a stock solution for producing initially encapsulated hemoglobin-containing liposomes by physical stimulation was obtained.

Example 2 The stock solution for producing initially encapsulated hemoglobin-containing liposomes by physical stimulation obtained in Example 1 was put into a syringe equipped with a nucleobor membrane filter with a pore size of 8 μm, and gas pressure (10 kg/d) was applied. , passed twice. This dispersion was added at 6 pm, 4 pm, 2 pm, and 1 pm.
, 0.8 μm%, 0.45 μm, and 0.2 μm in the same manner, and encapsulation was performed by extrusion method. (A part of the obtained dispersion was diluted and measured by semi-elastic light scattering method, and the average particle size was 6.
It was confirmed that a monolayer bilayer liposome of 5 nm±20 nm was obtained. ) The resulting dispersion was frozen in dry ice/methanol, then thawed in a 40°C water bath, and the encapsulation efficiency was improved by a freeze-thaw method. This dispersion was passed through a filter with a pore size of 0.4 μm at a pressure of 5 kg/cd, and the particle size was controlled by an extrusion method. At this stage, liposomes with an average radius of 7 Tnm±28 nm were obtained. The step of improving encapsulation efficiency using the freeze-thaw method and the particle size control step using the extrusion method were repeated five more times. By repeating this, the average radius L
A single N2 molecular membrane liposome system of 15 nm±15 nm was obtained. Next, by applying reduced pressure using a dialysis tube made of follower fiber with a pore size of 0.2 μm, the remaining unencapsulated hemoglobin and fractions with small particle sizes were removed and the solution was concentrated. Finally, the average radius of hemoglobin-containing liposomes was 130 nm ± 12
A hemoglobin-containing liposome was obtained with a final hemoglobin concentration of 5.1 wt% as determined by the cyanomet method and a lipid concentration of 6.2 wt% as determined by the molybdenum phosphate method.

(Theoretically calculated hemoglobin final concentration, i.e. (H
b) f = 8.5wt%, but the actual value was 5°1iit%. ) Example 3 Holding volume iL 8.3 (1/mol) (L
ipid (lipids)) r-5, swt% (Hb)
l = 15wt% (Hb) F = 6.7wt, % Hydrogenated egg yolk lecithin (H-EYL), cholesterol (
An alcohol mixed solution of phosphatidic acid (PA) was prepared. The composition is H-EYL/Chol=O
, T ~1.5, and H-EYL/PA=T~10.

Using the above mixed solution, hemoglobin-containing liposomes were prepared in the same manner as in Examples 1 and 2 except for the above. The pressure required when passing through the filter is 5.5% higher than the pressure in Example 2.
kg/cj is high, 15kg/ci! It cost. The obtained hemoglobin-containing liposome is H-EYL/Ch
For ol/PA (8:8:1), radius 15
0nm±23nm,) lb final concentration was 6.3wt%, and lipid concentration was 5.8wt%.

(Theoretically calculated hemoglobin final concentration, i.e. (H
b) F-6.7wt%, but the actual value was 6°3
It was 1%. ) Example 4 Retention capacity 6.4 (1/mol) (L) at 118 nm
ipid (lipids)) F = 5.8% 11t% (Hb
)i=15wt% (Hb)F=7.1%IIL% Diene type polymerizable lipid 1.2-bis(2,4-octadecajenoyl)-sn-glycero-3-phosphorylcholine (DODPC) 30g Hemoglobin-containing liposomes were prepared in the same manner as in Examples 1 and 2 except for the following. The hemoglobin-containing liposomes obtained had a radius of 118
nm±18 nm, final Hb concentration was 5.3 wt%, and lipid concentration was 8.5 wt%.

(Theoretically calculated hemoglobin final concentration, i.e. (H
b) F = 7.1wt%, but the actual value was 5°3wL%. ) 100 cc of this hemoglobin-containing liposome was placed in a quartz glass reaction tube and stirred. It was irradiated with ultraviolet light having a maximum absorption wavelength of 260 nm for 5 seconds and kept in the dark for - minutes. This was repeated, and the polymerized hemoglobin-containing polymerized liposome was obtained by scratching for 16 hours. There was no change in the particle size of the hemoglobin-containing polymerized liposome.

Example 5 30 g of egg yolk lecithin was dissolved in 100 cc of physiological saline solution, and this was dissolved in physiological saline solution 2 containing 45 g of hemoglobin.
When mixed with 00 cc (22.5 wL%), the same mixing ratio of egg yolk lecithin and hemoglobin as shown in Example 1 is obtained. This was then treated in the same manner as in Examples 1 and 2 to obtain hemoglobin-containing liposomes similar to those in Example 2.

Example 6 Retention volume: 14.0 (1/mol) (L) at 246 nm
ipid (fat'X) IF-5, 8wt% (Hb)
s = 22.5 wt% (Hb) F = 17.8 wt% 30 g of egg yolk lecithin was mixed with 300 cc (22.5 wt%) of a physiological saline solution containing 67.5 g of hemoglobin, and the mixture was mixed with a homogenizer under anoxic conditions for 4 hours. The mixture was treated at °C for 3 hours and uniformly dispersed and mixed to obtain a stock solution for producing initially encapsulated hemoglobin-containing liposomes by physical stimulation.

The stock solution obtained above for producing initial encapsulated hemoglobin-containing liposomes by physical stimulation was put into a syringe equipped with a nuclear membrane filter with a pore size of 8 μm, and gas pressure (10 kg/cd) was applied to it, and the mixture was allowed to pass through twice. Ta. This dispersion liquid is 6μm, 4μm, 2μm, 1μm, 0.
The mixture was sequentially passed through filters having pore sizes of 8 μm, 0.45 μm, and 10.2 μm to perform encapsulation by extrusion method. (A part of the obtained dispersion was diluted and measured by a monoculture light scattering method, and the average particle size was 65n.
It was confirmed that monolayer bilayer liposomes with m±20 nm were obtained. ) The resulting dispersion was frozen in dry ice and methanol, then thawed in a 40°C water bath,
We improved the encapsulation efficiency using the freeze-thaw method. 5 kg/c+ of this dispersion! The particles were passed through a filter with a pore size of 0.6 μm at a pressure of 0.6 μm, and the particle size was controlled by an extrusion method. At this stage, the average radius is 203 nm ± 61 nm.
obtained liposomes. The step of improving encapsulation efficiency using the freeze-thaw method and the particle size control step using the extrusion method were repeated five more times. By repeating this process, a monolayer bilayer liposome system with an average radius of 223 nm±41 nm was obtained.Then, by using a dialysis tube made of a follower fiber with a pore size of 0.2 μm and reducing the pressure, the remaining residual material was removed. The encapsulated hemoglobin and the small particle size fraction were removed (and the solution was concentrated.Finally,
The average radius of hemoglobin-containing liposomes is 246 nm±
At 21 nm, hemoglobin-containing liposomes were obtained with a final hemoglobin concentration of 17.8 iIIt% as determined by the cyanomet method and a lipid concentration of 5.8 wt% as determined by the molybdenum phosphate method.

(Theoretically calculated hemoglobin final concentration, i.e. (H
b) r = 23.4 wt%, but the actual value was 17
It was 8 wt%. ) Example 7 Retention capacity 12.2 (1/mol) at 218 nm (
Lipid (lipids) r=6.2wt% (Hb)
i=20. OwL% (Hbl F = 15.4wt%) 60g of DODPC is dispersed in 600d of pure water and sufficiently irradiated with ultrasonic waves (40 minutes at 60w) to obtain a liposome dispersion.

To this, 1.04 g of AAPD (water-soluble radical initiator: azobisamidinopropane dihydrochloride) was added, and after sufficient substitution with Nz gas, polymerization was carried out at 60° C. for 8 hours. The obtained poly-DODPC is a comb-shaped linear polymer in which only 2-acyl diene groups are polymerized and is soluble in organic solvents.

This was dissolved in alcohol and then freeze-dried to form a powder.

20 g of this powdered phospholipid was mixed with 200 cc (20 wt%) of a physiological saline solution containing 40 g of hemoglobin, and treated in an oxygen-free environment with a homogenizer at 4°C for 3 hours to uniformly disperse and mix, resulting in initial encapsulation by physical stimulation. A stock solution for producing hemoglobin-containing liposomes was obtained. This stock solution was put into a syringe equipped with a nuclear membrane filter with a pore size of 8 μm, and the gas pressure (10 kg/c
d) and passed twice. This dispersion liquid is 8 μm thick.
The mixture was sequentially passed through filters having pore sizes of 6 μm, 4 μm, 2 μm, 1 μm, and 0.6 μm to perform encapsulation by extrusion method. After freezing the obtained dispersion in dry ice/methanol,
It was thawed in a 0°C water bath and the encapsulation efficiency was improved by a freeze-thaw method. This dispersion was passed through a filter with a pore size of 0.6 μm at a pressure of 5 kg/cd, and the particle size was controlled by an extrusion method. At this stage, liposomes with an average radius of 181 nm±68 nm were obtained. The step of improving encapsulation efficiency using the freeze-thaw method and the particle size control step using the extrusion method were repeated five more times. By repeating this process, the particle size distribution narrowed as the average radius increased, yielding a monolayer bilayer liposome system with a radius of 2 Qenm±41 nm. Next, by applying reduced pressure using a dialysis tube made of follower fiber with a pore size of 0.2 μm, the remaining unencapsulated hemoglobin and a fraction with a small particle size were removed, and the solution was contracted. The average radius of the final hemoglobin-containing liposomes is 218n.
m±36 nm, a hemoglobin-containing liposome was obtained with a final Hb concentration of 15.4 wt% as determined by the cyanomet method and a lipid concentration of 6.2 wt% as determined by the molybdenum phosphate method.

(Theoretically calculated hemoglobin final concentration, i.e. (H
b) F = 19.3d%, but the actual value was 15°4wL%. ) The obtained liposome was placed in a quartz glass reaction tube and irradiated with ultraviolet light for 5 minutes under Nz to cause a crosslinking reaction between l-acyl chain diene groups. In this way, dynamically stable hemoglobin-containing liposomes were obtained.

There was no change in particle size.

〔Effect of the invention〕

The present invention has the configuration as described above. Efficient encapsulation has become possible because the minimum amount of lipids, mainly composed of phospholipids, necessary for encapsulating hemoglobin in a monolayer bilayer membrane, is incorporated into hemoglobin.

Because it is an ultra-thin monolayer bilayer membrane, the liposome contains only a small amount of lipids mainly composed of phospholipids, resulting in low viscosity, good blood circulation dynamics, and extremely low toxicity. The initial encapsulated stock solution for producing hemoglobin-containing liposomes is subjected to an encapsulation process using an extrusion method, a process to improve encapsulation efficiency using a freeze-thaw method, and a particle size control process using an extrusion method, making it possible to make capsules highly homogeneous. It became. Therefore, according to the present invention, an encapsulating material having a structure similar to that of lipid xi, which is primarily composed of phospholipids originally derived from living organisms, is used, and moreover, it is an ideal capsule with an ultra-thin monolayer bilayer membrane. Since the toxicity is extremely low, the viscosity is low, and the capsule is highly homogenized, it is possible to industrially provide hemoglobin-containing liposomes that serve as ideal artificial red blood cells.

Claims (3)

[Claims] (1) Calculate the amount required to form a monolayer bimolecular membrane of lipids whose main component is phospholipid using formula 1: W=4π[r^2+(r-Δd)^2]・N/N_A・
S [where, W is the amount of lipids mainly composed of phospholipids (mol/l), r is the capsule radius (Å), Δd is the capsule membrane thickness (Å), and N is the capsule concentration (mol/l). ), N
_A is Avogadro's number (6.02 x 10^2^3), S is the average value of the molecular occupied area of lipids whose main component is phospholipid (Å^2)], and the phospholipid is the main component. The amount of hemoglobin that can be contained in a liposome formed in the amount necessary to form a monolayer bilayer membrane of lipids is calculated using formula 2 [Hb]_i=[Hb]_f/{4/3π( r−Δd)
^3N} [However, [Hb]_i is the concentration (g/l) of Hb (hemoglobin) charged at the time of capsule preparation, [Hb]
_f is the final Hb concentration (g/l), r is the capsule radius (Å
), N is the capsule concentration (capsules/l)], and the amount of lipids mainly composed of phospholipids determined by the above calculation formula 1 is uniformly dispersed and mixed with the amount of hemoglobin determined by the above calculation formula 2. A hemoglobin-containing liposome encapsulated in a monolayer bilayer membrane of lipids mainly composed of phospholipids, which is produced using a stock solution for producing hemoglobin-containing liposomes that are initially encapsulated by physical stimulation. (2) Calculate the amount required to form a monolayer bimolecular membrane of lipids whose main component is phospholipid using formula 1: W=4π[r^2+(r-Δd)^2]・N/N_A・
S [where, W is the amount of lipids mainly composed of phospholipids (mol/l), r is the capsule radius (Å), Δd is the capsule membrane thickness (Å), and N is the capsule concentration (mol/l). ), N
_A is Avogadro's number (6.02 x 10^2^3), S is the average value of the molecular occupied area of lipids whose main component is phospholipid (Å^2)], and the phospholipid is the main component. The amount of hemoglobin that can be contained in a liposome formed in the amount necessary to form a monolayer bilayer membrane of lipids is calculated using formula 2 [Hb]_i=[Hb]_f/{4/3π( r−Δd)
^3N} [However, [Hb]_i is the concentration (g/l) of Hb (hemoglobin) charged at the time of capsule preparation, [Hb]
_f is the final Hb concentration (g/l), r is the capsule radius (Å
), N is the capsule concentration (capsules/l)], and the amount of lipids mainly composed of phospholipids determined by the above calculation formula 1 is uniformly dispersed and mixed with the amount of hemoglobin determined by the above calculation formula 2. Phospholipids are produced by subjecting a stock solution for producing hemoglobin-containing liposomes that is initially encapsulated by physical stimulation to an encapsulation process using an extrusion method, an encapsulation efficiency improvement process using a freeze-thaw method, and a particle size control process using an extrusion method. A method for producing a hemoglobin-containing liposome encapsulated in a monolayer bilayer membrane of lipids containing as a main component. (3) Calculate the amount necessary to form a monolayer bimolecular membrane of lipids whose main component is phospholipid using formula 1: W=4π[r^2+(r-Δd)^2]・N/N_A・
S [where, W is the amount of lipids mainly composed of phospholipids (mol/l), r is the capsule radius (Å), Δd is the capsule membrane thickness (Å), and N is the capsule concentration (mol/l). ), N
_A is Avogadro's number (6.02 x 10^2^3), S is the average value of the molecular occupied area of lipids whose main component is phospholipid (Å^2)], and the phospholipid is the main component. The amount of hemoglobin that can be contained in a liposome formed in the amount necessary to form a monolayer bilayer membrane of lipids is calculated using formula 2 [Hb]_i=[Hb]_f/{4/3π( r−Δd)
^3N} [However, [Hb]_i is the concentration (g/l) of Hb (hemoglobin) charged at the time of capsule preparation, [Hb]
_f is the final Hb concentration (g/l), r is the capsule radius (Å
), N is the capsule concentration (capsules/l)], and the amount of lipids mainly composed of phospholipids determined by the above calculation formula 1 is uniformly dispersed and mixed with the amount of hemoglobin determined by the above calculation formula 2. Stock solution for the production of initially encapsulated hemoglobin-containing liposomes by physical stimulation.