JPS5849311A - Preparation of stable liposome - Google Patents

Abstract

PURPOSE:To prevent the destruction of liposome caused by the attack with the phospholipase in the living body, and to enable the selective and concentrated transfer of drug to the aimed organ, by covering the surface of a liposome membrane with an ester of polysaccharide and fatty acid. CONSTITUTION:The surface of a single-layered or multi-layered liposome membrane composed of only a lipid (e.g. phosphatidylcholine) or of lipid and sterol, is coated with the ester of a polysaccharide (e.g. dextran, amylopectine, pullulan, chitosan, etc.) and fatty acid (e.g. lauric acid, myristic acid, palmitic acid, stearic acid, etc.). The coating can be carried out by adding an aqueous solution containing said ester to an aqueous solution containing liposome, and stirring the mixture.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing stable liposomes in which the surface of the liposome membrane is coated with an ester of polysaccharide and fatty acid.

As interest in so-called organ-directed preparations gradually increases in the medical and pharmaceutical fields, technology that utilizes liposomes has come to be seen as a means to achieve this goal. In other words, we focused on the fact that liposomes exist as unique subosomes in each organ.

Techniques are being developed that aim to selectively and intensively deliver a drug to a target organ by containing a drug in a liposome and administering the drug.

Means for utilizing liposomes as drug carriers have been known for a long time, 9 for example, JP-A-52-143218, q!
i Kaisho 52-151718. Japanese Patent Publication No. 53-133616
．． The invention in Japanese Patent Publication No. 55-8488 can be mentioned. All of these inventions relate to techniques for holding specific biological components or pharmaceutical components in liposomes as carriers.

Eventually, these technologies will be applied to other biocomponents in medical and pharmaceutical sciences.
It is expected that it will be applied to pharmaceutical ingredients.

However, one problem with this technology is 2.
The following issues have been pointed out in the past. That is,
Liposomes containing biological or pharmaceutical components are destroyed by in-vivo phospholipase attack before they reach the target organ.

This means that the contents will be released. In fact, it is observed that the release of the contents due to the destruction of the liposomes is slow, as in the control samples of the Examples described below. Therefore, it is necessary to provide a means that can prevent the destruction of liposomes due to attack by in vivo phospholipases by an appropriate method, and furthermore, it is necessary to provide a means that can rapidly release the contents in the target organ. However, it is desirable to appropriately control both , and slow rates. In other words, in order for this technology to be practically completed in the field of medicine and pharmacy, 2. There is a need to provide a means by which liposomes themselves can be stably maintained during in-vivo processes and the release of the contents in target organs can be controlled in a planned manner as necessary.

In view of this situation, the present inventors developed liposomes.

In particular, we investigated a method to protect phospholipids in liposomes from external attack, and as a result, we discovered that the desired purpose could be achieved by coating liposomes with an ester of polysaccharide and fatty acid. completed.

Therefore, the present invention is a method for producing stable liposomes characterized by being coated with an ester of polysaccharide and fatty acid.

The present invention will be explained below.

In the present invention, liposomes refer to unilamellar and multilamellar liposomes composed of lipids, particularly phospholipids alone or phospholipids and sterols, and are prepared by conventionally known methods for producing liposomes. Therefore, for example, liposomes obtained by preparing a thin film of egg yolk lecithin in an eggplant-shaped flask, placing it in glass beads and peeling it off to form a liposome film are included.

The content substance carried by the liposome may be either water-soluble or fat-soluble. However, water-soluble substances are accommodated in the lumen of the liposome, and fat-soluble substances are accommodated in the liposome II[K.

Next, the polysaccharide according to the present invention refers to a polysaccharide having a molecular weight of 40,000 or more, and specifically includes dextran, airopectin, pullulan, dextran II*, chitoic acid, pullulan sulfate, and the like. In the present invention, polysaccharides have two different molecular weights! A plurality of polysaccharides may be used in combination, such as a combination of TI or higher polysaccharides, or a combination of amylopectin and dextran.

Furthermore, the fatty acids according to the present invention include lauric acid, myristic acid,
A combination of one to four selected from the group consisting of valmitic acid and stearic acid. When a liposome is coated with the ester according to the present invention, it is estimated that the alkyl chain of the -2 fatty acid is oriented in the *m direction in the lipid layer of the liposome. Therefore, the above fatty acids are particularly preferred as
This is believed to be because the length of the alkyl chain is suitable for wedge-shaped orientation into the liposomal lipid layer.

Next, the production of an ester of a polysaccharide and a fatty acid can be carried out roughly as follows. First, a polysaccharide is dissolved in anhydrous dimethylformamide by heating at 60 to 70°C. A pre-dissolved fatty acid chloride solution is added to anhydrous pyridine and anhydrous dimethylformanad, and the mixture is stirred at 60 to 10° C. for several hours and then at room temperature for 24 hours. After the reaction is complete, add ethanol to the reaction mixture and a white precipitate will form.
This is taken off as P, washed with ethanol, further dispersed in ether, and taken off again as R. Dry under reduced pressure to obtain the desired ester.

The ester obtained here has an IR spectrum (KBr+
method), n'-NMB spectrum (solvent d@-〇MSO,
The degree of fatty acid substitution can be determined in addition to the internal standard TM8).

The degree of fatty acid substitution refers to the number of fatty acids introduced per 100 sugar units, and this is measured by 5KH'-NMR as shown below. For example, when a valmitoyl group is introduced, the amount of introduction is 0.0% in the H'-NMR spectrum.
The peak areas of protons of valmitoyl group appearing at 9 ppm and 1.28 ppm and 3.5 to 5.2 ppm
It is given by the ratio of the peak area of protons in bran to K3J. That is, when X valmitoyl groups are substituted in sugar unit 10011m, the number of protons of the sugar is 9x+10 (100-x), and the number of protons of the valmitoyl group is 31x. Therefore H' −N
MR The number of sugar protons, y, determined from the integral curve of the R spectrum.

Also, assuming that the number of protons in the valmitoyl group is 2.

It is 1.000z. When the degree of substitution obtained in this way was measured for the ester used in the present invention, it was found that a lower degree of substitution gave a preferable result.2For example, 0. Cor 5 is sufficient.

Furthermore, in order to coat liposomes with the ester according to the present invention, the ester-containing aqueous solution may be added to the aqueous solution in which the liposomes are formed and stirred.

For coating, it is sufficient to coat a single ester, but
Two or more types of esters according to the present invention may be combined to form a so-called composite ester of 1 to 1 type.

The present invention and the effect of the present invention in stabilizing liposomes will be explained in more detail with the following Examples.

1! Example 1 6-carpoxyfluorescein (hereinafter abbreviated as 6-CF) was used as a water-soluble substance supported on a sample liposome.

This 6-CF does not emit a silicon source due to concentration quenching at high concentrations. Therefore, a high concentration (200mM) of 6-CF
The leakage of 6-OF from the internal aqueous phase of the liposome to the external aqueous phase can be investigated by observing the increase in light emission when 6-OF is trapped in the internal aqueous phase of the liposome.

Next, a liposome containing 6-CF in the internal aqueous phase was prepared as follows.

3011 g of egg yolk lecithin was added to a 50 iu eggplant-shaped flask together with 2111 chloroform, and after completely dissolving, the solvent was completely removed under reduced pressure using a rotary evaporator to prepare a thin layer of egg yolk lecithin.

This was dried in a vacuum desiccator overnight to completely remove chloroform. Next, put several Olas beads into this flask and add 200mM 6-CF aqueous solution (pH 8,
After adding 2117 (adjusted with 6Na0B).

The thin film was peeled off with a Vort@x 岨过・r to form a ribonome gauze. This operation was repeated twice to obtain a total of 4 dk dispersed. Next, under a nitrogen atmosphere on water, ultrasonic treatment (25We
(30 seconds stop every minute) was carried out for 20 minutes. In the liposomes obtained here, five types of esters a as shown in Table 1 are added.
Coated with PJe.

Table 1 The coating was performed as follows. 6-CF solution (200mM pH 8,
6) An aqueous ester solution (30~/d) td was added to 4i1 and stirred with a stirrer at 20°C for 1 hour.

Next, add this to S@pharos@4 B column (fIi1
8m x 600m) with 200mM NaCj, 20mM
Tris-HCl buffer (pH 8,6°)
Gel filtration was performed using this as a developing solvent.

The dropping rate of the solvent was set to 1 drop every 5 seconds, and up to a flux W/40 was collected. Approximately 4-120 drops per unit of flaxin
Met. The elution curve was obtained by tracking the elution process using an ultraviolet-visible spectrophotometer for the absorption of 6-CF at 510 nm. From this, fuchsia containing multiple layers and one buffy coat liposome was collected. The elution curve is shown in Figure 1k.

Using the coated liposome-containing aqueous solution prepared in the above manner as a test sample, changes over time in 6-CF flowing out from the liposome internal aqueous phase were determined by the method described below. As a control sample, a sample obtained by performing the gel filtration described above before coating was used.

Method (1) First, the efflux of 6-CF was measured over time when a liposome membrane was coated with ester 30 da of egg yolk lecithin 30119. Measurements were carried out at 50° C. and the increase in fluorescence at 520 nm of 6-C11′ -x was followed (←470 nm).

The amount of 6-CF trapped in the aqueous phase inside the liposome is 1111 in the liposome solution with lQ vo1% Tr.
The liposome membrane was broken by adding 304 times of 1tot+X-100 aqueous solution, and the fluorescence intensity at 520 nm of 6-CF at that time was determined. The value of this fluorescence intensity was set as 100, and the increase in fluorescence intensity due to the outflow of 6-CF was calculated in units of increase.

(2) Next, the effects of ester concentration on the outflow of 6-CF were investigated using the esthetics bath described in Table 1.

(In this experiment, egg yolk lecithin 309 was coated with 60, 30 to 10, 10 μg of cyte ester a.) The results are shown in FIGS. 2 to 4. Figure 2 shows the 6-CFf) outflow when the liposome is a multilamellar liposome; Figure 3 shows the 6-CFf) outflow when the liposome is a unilamellar (one buffy coat) liposome; The marked line is ester a, the Δ marked line is ester b, and the mu marked line is ester C2.
The marked lines are for specimen samples each coated with ester 6.

From FIGS. 2 and 3, it has been found that by coating a liposome membrane with the ester according to the present invention, the release of the contents can be appropriately controlled to be rapid or slow.

Figure 4 is a diagram showing the release of 6-CF when the liposome is a multilayered liposome coated with ester 1. In Figure 9, the 0-marked line is a control sample, and Muinwa's ester amount is 2% compared to egg yolk lecithin. For each specimen sample, the Δ line is 1 times, and the * mark line is 1i3 times. From FIG. 4, it was found that the release of the contents could be appropriately controlled by adjusting the ester content in the range of 10 to 30 Da compared to the egg yolk lecithin of 30 Da.

Example 2 (Stabilization against phospholipase attack) Sample In the description of the sample in Example 1, buffer (
An aqueous solution containing coated liposomes (1 buffy coat liposome) was prepared as a test sample by the same method as described in the same section except that p)j8,6)2iu was added. Ester 1 listed in Table 1 was used as the ester, and the amount added was 0.5y/3.2su per egg yolk lecithin of 3.4 x 10-'M (0.26/m)K. In addition, as a control sample, a sample obtained by directly performing gel filtration without performing coating treatment in the preparation of the above-mentioned specimen sample was used.

Method and Quantification Samples were heated at 37°C, pH 8.0 (200mM Tri@
Phospholipase-○ (abbreviated as PLD) was added thereto, and the state in which egg yolk lecithin was hydrolyzed by PLO and the liposomes were destroyed was monitored over time. For the Il constant, add sodium 8-atelino-1-naphthalenesulfonate (abbreviated as ANS) to the sample as an indicator in advance, and then
The fluorescence intensity in nm was measured over time. That is, ANS is a substance that hardly emits fluorescence in an aqueous solution, but when liposomes are present in a bathing liquid, it is adsorbed to the membrane surface and emits strong fluorescence.

Therefore, if liposomes are destroyed by PLD, the ANS
is released from the membrane surface into the water, and the fluorescence intensity decreases. This phenomenon was utilized to perform the -j determination as described above.

result The results are shown in FIG. In the figure, the border marked with 0 is the control sample.

・The marked lines indicate those in the specimen sample. As is clear from FIG. 5, the decrease in fluorescence intensity of the ester-coated liposomes is suppressed compared to that of the uncoated liposomes. It turns out that this is because coating the liposome surface with ester protects the egg yolk lecithin from PLD attack.

[Brief explanation of the drawing]

FIG. 1 is a drawing corresponding to FIG. 1K described in Example 1, and shows the elution curve in gel filtration. 2 and 3 are drawings corresponding to FIGS. 2 and 3 described in Example 1, and show the outflow of 6-CF in each case where the liposome is a multilamellar liposome and a unilamellar liposome. FIG. 4 is a diagram corresponding to FIG. 4 described in Example 1, and shows the outflow of 6-CF when the liposome is a multilayer liposome coated with ester a. FIG. 5 is a diagram corresponding to FIG. 5 described in Example 2, and shows a situation where the fluorescence intensity decreases as a result of liposome destruction by phospholipase-○. Figure 1 fraction number Time (+nin) 213 Figure 4 ρ lQ −IJ 3ρ procedural correction *(
Voluntary) July 27, 1980 Haruki Shimada, Commissioner of the Japan Patent Office1, Indication of the case, Patent Application No. 147587, filed in 19822, Title of the invention: Process for producing stable liposomes3, Relationship with the amended person case Patent Applicant name (021) Eisai Co., Ltd. (1) Column for detailed explanation of the invention in the specification (2) Full text of the specification 5, contents of amendments (1) ``0M80.J'' on the 4th line from the bottom of No. 11 (2) Copying of the specification (no changes to the contents other than (1) above)

Claims (6)

[Claims] (1) A method for producing stable liposomes, which comprises coating the surface of the liposome membrane with an ester of polysaccharide and fatty U5 acid. (2) Polysaccharides are dextran and amylopectin. The method for producing stable liposomes according to claim 1, which is a combination of 1 to 6 cypresses selected from the group consisting of pullulan, dextran sulfate, chitic acid, and pullulan sulfate. (3) 1 to 4 in which the fatty acid is selected from the group consisting of lauric acid, myristic acid, noclumitic acid, and stearic acid;
A method for producing a stable liposome according to claim 1 or 2, which is a combination of 11i. (4) The method for producing stable liposomes according to claims 1 to 3, wherein the liposome membrane is a liposome membrane composed of lipid or lnI substance and sterol. (5) The method for producing a stable liposome according to claim 4, wherein the potassium substance is phosphatidercholine. (6) The method for producing stable liposomes according to claim 5, wherein the ester of pullulan and valmitic acid is 1/3 to 1 part by weight per 1 part by weight of phosphatidercholine.