JPH0518807B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel liposome containing an active substance that has high mechanical strength and high safety in vivo. Liposomes are composed of two molecular layers of phospholipids such as phospholipids and cholesterol, and have a composition similar to that of cell membranes, making them suitable for creating highly safe artificial cells, and research is being conducted on artificial blood using them. There is. However, conventional liposomes are unstable and break easily, making it difficult to bind and encapsulate proteins such as hemoglobin. Furthermore, even if hemoglobin, which has an oxygen-transporting function, is encapsulated in liposomes, there have been few reports on its rheology, such as its deformability, and it was unclear whether the liposomes would be suitable for clinical use. The present inventors have conducted extensive research in view of the above problems, and have found that polysaccharides with D-glucosamine as the main skeleton component, such as carboxymethyl chitin and chitosan, are added to the bimolecular layer of phospholipids that form the wall of liposomes. It has been found that by mixing or binding, liposomes can be obtained that have high mechanical strength and exhibit flow characteristics similar to those of mammalian blood such as sheep hemoglobin and human hemoglobin. That is, the present invention is characterized by an active substance-containing liposome comprising a bimolecular layer of phospholipid mixed with or bound to a polysaccharide whose main skeleton component is D-glucosamine as a mechanical reinforcement. According to the present invention, since it has higher mechanical strength than conventional liposomes, it not only has high safety in and outside of the body, but also has a high encapsulation rate. Due to its presence, it has chemical reactivity,
Chemical modifications can also be made to impart cell specificity. The phospholipid used in the active substance-containing liposome of the present invention is not particularly limited as long as it is an amphiphilic lipid that has been conventionally used for forming the wall of liposomes, such as lecithin, kephalin, plasmalogen, α-glyceryl ether, Examples include inositol phospholipids, aminoacylphosphatidylglycerose, sphingomillien, etc. These may be used alone or in combination, and sterols such as cholesterol and ergosterol may be added as necessary. . In addition, polysaccharides having N-acetyl-D-glucosamine as a main skeleton component to be mixed or bonded to the above-mentioned phospholipids as a mechanical strengthening agent include, for example, carboxymethyl chitin, carboxyethyl chitin, which are derivatives of chitin, and hydrated chitin. There are chitosan obtained by decomposition, carboxymethyl chitosan, hyaluronic acid in which N-acetyl-D-glucosamine and D-glucuronic acid are alternately bonded, and its derivatives, and these can be used alone or in an appropriate combination of two or more types. be able to. When forming liposomes using the polysaccharide as the mechanical reinforcement, the polysaccharide is contained in the wall of the liposome in an amount of 0.01 to 0.2% by weight,
Preferably it is present in an amount of 0.02 to 0.05% by weight. If the polysaccharide content in the wall membrane exceeds 0.2% by weight, it will coalesce, and if it is less than 0.01% by weight, stable liposomes with excellent mechanical strength cannot be obtained. On the other hand, active substances to be encapsulated (clathrated) in the liposomes of the present invention include mammalian red blood cells such as sheep hemoglobin and human hemoglobin, which are the main components of artificial blood, synthetic hemoglobin substitutes, insulin, oxytocin, and pasopressin. , heptide hormones such as glucagon and corticotropin, steroid hormones such as progesterone and adrenal hormone, antibiotics such as penicillin, streptomycin, chloramphenicol and pyomycin, urease, pepsin, trypsin,
Enzymes such as arginase, vitamins such as thiamine, nicotinamide, ascorbic acid, and 5 others
-Medicinal agents such as FU (5-fluorouracil) and mitomycin can be mentioned. To prepare the liposome of the present invention which includes the above-mentioned active substance and has high mechanical strength, a phospholipid such as lecithin is dissolved in an organic solvent such as dichloromethane, chloromethane, or benzene, and this is dissolved in the above-mentioned sheep hemoglobin, etc. The mixture is mixed with an aqueous solution of the active substance and stirred vigorously to form a W/O emulsion. In this case, the appropriate ratio of the organic solvent to the phospholipid is 1.0 to 10.0 parts by weight of the phospholipid per 100 parts by volume of the organic solvent. Further, the concentration of the aqueous solution of the active substance is suitably 5.0 to 50.0% (W/V). A solution prepared by dissolving or dispersing the polysaccharide as a mechanical strengthening agent in an aqueous medium is added to this emulsion and stirred vigorously to form a W/O/W type emulsion, which is then further stirred to form a W/O/W type emulsion. completely evaporate. Examples of the aqueous medium in which this polysaccharide is dissolved or dispersed include water, physiological saline,
PH regulator (sodium hydroxide, potassium hydroxide),
Buffers (e.g. Tris-HCl aqueous solution, phosphoric acid 1
sodium, disodium phosphate aqueous solution). In this case, the appropriate concentration of the polysaccharide in the aqueous medium is 0.05 to 0.5% (W/V), and when adding it to the above W/O type emulsion, the entire amount may be added at once or this amount may be added to the W/O type emulsion. may be added in several portions. Note that these series of operations are
It is preferably carried out at room temperature, and in an inert atmosphere such as nitrogen, especially when the active substance is isolated from a living body, such as sheep hemoglobin or human hemoglobin. Thus, stable liposomes containing active substances and protected by polysaccharides are dispersed in W/O/
A W-shaped emulsion is obtained. This liposome has particles concentrated in the 140-420nm range with an average diameter of
It has a spherical shape with a wavelength of 310 nm and can freely pass through capillaries, and can be easily separated by centrifugation at 2,000 to 12,000 g for 5 to 30 minutes. Therefore, this emulsion can be used as it is, but after separating and removing the active substance that was not encapsulated in the liposomes by the above-mentioned centrifugation etc., for example, in the case of hemoglobin-included liposomes, it can be used as is or if necessary, it can be used as is. It can be used as artificial blood that can be transfused together with nutritional supplements, as well as other desired medicines such as injections, oral preparations, suppositories, and cosmetics. Next, the present invention will be specifically explained using examples. Example 1 Carboxymethyl chitin (manufactured by Nanyo Kasei Co., Ltd.)
was dissolved in water to make a 0.2% (W/V) solution.
Dextran (Mw73300 Sigma, Chemical Company St. Louis) was dissolved in Tris-HCl buffer (PH7.4) to form a 6% (W/V) solution. Egg yolk lecithin (manufactured by Asahi Kasei Co., Ltd.) was dissolved in dichloromethane in a nitrogen stream to prepare a solution with a concentration of 50 mg/ml. Sheep red blood cell lysate was prepared by osmotic hemolysis. 10 ml of the lecithin solution was added to 10 ml of the above lysed sheep blood and stirred vigorously for 1 minute using a magnetic stirrer to prepare a W/O emulsion. This emulsion was quickly added to 100 ml of carboxymethyl chitin while stirring at 313 rpm to obtain a W/O/W type emulsion. After 10 minutes while continuing to stir,
Furthermore, add 100ml of carboxymethyl chitin aqueous solution,
Continue stirring until dichloromethane completely evaporates, and add ovine hemoglobin clathrate liposomes (hereinafter referred to as
(simply called ARBC). During this operation, the temperature was kept at room temperature. The resulting aqueous dispersion was centrifuged at 12,000 g for 1 hour to collect ARBC. After washing this ARBC with distilled water, it was redispersed in Tris-HCl buffer (PH7.4 ionic strength = 0.154) to a particle concentration of 10, 20, 30, and 40% (W/V), respectively.
ARBC dispersion was prepared. In addition, a sheep red blood cell (hereinafter simply referred to as SRBC) dispersion liquid (PH7.4) was prepared in the same manner. The flow properties of the ARBC and SRBC suspensions were measured using a concentrated cylindrical rotational viscometer (LS-100, Contraves Industrial Products Ltd.) equipped with a control device (Rheomart 30, Contraves Industrial Products Ltd., Switzerland). The amount of suspension used in the measurement was 1 ml, and the temperature was kept constant at 25±0.5°C. Scanning electron microscopy of the obtained ARBC (×5000)
As a result of observation, ARBC was found to be perfectly spherical with an average particle size of 310 nm. These blood cells can therefore freely pass through the capillaries. FIG. 1 shows the flow characteristics when the concentration of the ARBC suspension was changed. In addition, Figure 2 shows ARBC and SRBC suspensions (PH7.4) with particle concentration as a variable.
The specific viscosity of The specific viscosity of SRBC suspensions increases significantly with particle concentration. In contrast, the present invention
ARBC increases linearly over the experimental range. That is,
The relationship between specific viscosity and particle concentration of SRBC suspension agrees well with Brinkman's equation, but
The specific viscosity of ARBC suspension has a slope of approximately 2.3
Follow Einstein's formula. On the other hand, it is known that the flow characteristics of blood and red blood cell suspensions follow the Casson plot. FIG. 3 shows a Katson plot of the above ARBC suspension, and it can be seen that the ARBC suspension exhibits linearity. FIG. 4 shows the flow characteristics of an ARBC suspension in a Tris-HCl buffer solution containing 6% (W/O) dextran. Here, dextran is used as a blood substitute or thickening agent. Comparing FIG. 4 and FIG. 1, it can be seen that the flow resistance of the ARBC suspension with dextran is higher at low shear rates than the ARBC suspension without dextran. Figure 5 also shows ARBC and SRBC when dextran concentration is used as a variable.
The specific viscosity of a 40% (V/V) suspension is shown. ARBC
The specific viscosity of the suspension decreases with increasing dextran concentration, as in the case of SRBC suspensions. At a dextran concentration of 8% (W/V), the specific viscosity decreases by 20% compared to that without dextran. This indicates that the flow properties of ARBC and SRBC are significantly affected by the presence of dextran. Moreover, FIG. 6 shows the relationship between the mixing ratio and specific viscosity of a mixed suspension of ARBC and SRBC. The total particle concentration of this mixed suspension was kept constant at 40%. As the dispersion medium, there are those that use dextran and those that do not. When the mixing ratio of ARBC to SRBC is increased, the specific viscosity of the suspension tends to decrease regardless of the dextran concentration. As the proportion of ARBC decreases, the specific viscosity approaches that of SRBC suspension (PH7.4). Thus, the flow properties of the suspension of ARBC (PH 7.4) according to the invention are pseudoplastic and exhibit the same behavior as the SRBC suspension. The specific viscosity of both ARBC and SRBC suspensions increases with particle concentration in the dispersion medium. However, the relationship between specific viscosity and particle concentration for ARBC suspensions matched well to Einstein's equation, whereas for SRBC suspensions it fit Brinkman's equation. This is considered to be because the carboxymethyl chitin added as a mechanical strengthening agent is adsorbed on the surface of ARBC by ionic bonding. On the other hand, a mixed suspension of ARBC and SRBC (PH7.4)
Regarding the flow characteristics of , it can be seen that the interaction between ARBC and SRBC weakens as the mixing ratio of ARBC increases. Even if dextran is added to the dispersion medium, its fluidity properties are hardly affected. Therefore, it is inferred that ARBC in a mixed suspension reduces particle-to-particle interaction due to the strong hydration power of carboxymethyl chitin. Example 2 Add 20 ml of lecithin in chloroform solution to 20 ml of 20% human hemoglobin solution and mix with a magnetic stirrer for 1 hour.
Stir vigorously for a minute to obtain a W/O type emulsion. An aqueous solution of 0.3% chitosan in this emulsion.
Add 100ml quickly and stir at 400rpm. After another 10 minutes, add 200 ml of carboxymethyl chitosan and continue until the chloroform evaporates. Note that this series of operations is performed at room temperature. The resulting liposomes have the same oxygen transport function as human red blood cells. Example 3 1 part of a dichloromethane solution containing 30 mg/ml lecithin and a small amount of cholesterol and 1 part of a 40 U/ml insulin aqueous solution are emulsified to form a W/O emulsion. Next, this emulsion is quickly added to a 0.02% (W/V) carboxymethyl chitin aqueous solution in a stirring system to form a W/O/W type emulsion. Thereafter, the temperature is raised to evaporate dichloromethane to form insulin-encapsulated liposomes. Example 4 2 parts of 50mg/ml lecithin benzene solution and 50mg/ml
Mechanically emulsify 1 part of albumin aqueous solution of W/
Let it be an O-type emulsion. This emulsion was dispersed in a 0.1% (W/V) chitosan aqueous solution in a stirring system to form a W/O/W type emulsion, and then the temperature of the system was raised to evaporate benzene to form albumin-encapsulated liposomes. Example 5 Preparation of carboxymethyl chitinated liposome containing glucose First, a 0.01 to 0.5% (W/V) carboxymethyl chitin aqueous solution is prepared. A W/O emulsion is prepared by mixing 1 part of the glucose solution of the inclusions and 1 part of a 50 mg/ml egg yolk lecithin dichloromethane solution and stirring. This emulsion was quickly added to 200 parts of each of the above carboxymethyl chitin aqueous solutions and stirred to form a W/O/W type emulsion. All previous operations are performed at room temperature. Next, when the temperature of the system is raised to 40°C, dichloromethane evaporates from the emulsion. In this process, the lecithin that makes up the liposome,
Electrostatic bonding with carboxymethyl chitin occurs. Furthermore, when dichloromethane is completely evaporated, carboxymethylated liposomes can be obtained. The table below shows the rate of glucose incorporation into the liposomes for each carboxymethyl chitin concentration when glucose-containing liposomes were prepared according to the above method. 【table】

[Brief explanation of drawings]

The drawings show one embodiment of the present invention, in which Figure 1 is a flow characteristic diagram when the concentration of the ARBC suspension according to the present invention is varied, and Figure 2 is a diagram showing the above-mentioned flow characteristics with the particle concentration as a variable.
A graph showing the specific viscosity of ARBC and SRBC suspension (PH7.4), Figure 3 is a graph showing the Katson plot of the above ARBC suspension, and Figure 4 is 6% (W/
Figure 5 shows the flow characteristics of ARBC suspension in Tris-HCl buffer solution containing dextran (O). Figure 5 shows ARBC when dextran concentration is a variable.
FIG. 6 is a graph showing the relationship between the mixing ratio and the specific viscosity of a mixed suspension of ARBC and SRBC.

Claims (1)

[Scope of Claims] 1. An active substance-containing liposome comprising a bimolecular layer of phospholipid mixed with or bound to a polysaccharide having D-glucosamine as its main skeleton component. 2. The active substance-containing liposome according to claim 1, wherein the polysaccharide is one or a mixture of two or more selected from the group consisting of carboxymethyl chitin, carboxymethyl chitosan, and chitosan. 3. The active substance-containing liposome according to claim 1, wherein the polysaccharide is hyaluronic acid and/or a derivative thereof.