KR20080016576A - Method of producing liposomes containing gas enclosed therein - Google Patents

Abstract

It is intended to provide liposomes containing a gas enclosed therein which are useful in ultrasonic diagnostics and ultrasonic therapies. A method of producing liposomes containing a gas enclosed therein characterized by comprising filling the space in a sealed container, which contains a liposome suspension in a volume amounting to 20 to 80% of the inner capacity thereof, with a fluoride gas or a nitrogen gas and then conducting an ultrasonic treatment.

Description

METHOD OF PRODUCING LIPOSOMES CONTAINING GAS ENCLOSED THEREIN}

The present invention relates to gas-sealed liposomes useful as ultrasonic diagnostic agents and ultrasonic therapeutic agents, and methods for their preparation.

Ultrasonic diagnostic devices are used in a wide range of obstetrics, gynecology, urology, and the like. On the other hand, as an ultrasonic imaging agent, the method of imaging and diagnosing by ultrasonic irradiation after administering gas containing microparticles (micro bubble) is developed (refer patent documents 1-5).

However, since the conventional microbubble contains gas in the large particles formed of albumin, the particle diameter is large, 2 to 6 µm, and the transferability to the deep structure is low.

[Patent Document 1] U.S. Patent # 4572205

[Patent Document 2] US Patent No. 4718433

[Patent Document 3] U.S. Patent No. 4774958

[Patent Document 4] US Patent No. 4844852

[Patent Document 5] US Patent No. 4957656

Disclosure of the Invention

Problems to be Solved by the Invention

It is therefore an object of the present invention to provide a method for producing a stable microbubble having a small particle diameter by simple operation, a microbubble obtained in this way, and a method of using the same.

Means to solve the problem

Therefore, as a result of various studies, the inventors have made a liposome prepared in advance as a suspension, and added it to a sealed container having a constant porosity, and filled the fluoride gas or nitrogen gas in the void portion, and then subjected to ultrasonic treatment. It was found that this small gas-sealed liposome can be stably prepared. Moreover, since the particle diameter of the gas encapsulated liposome obtained in this way depends on the particle diameter of the previously prepared liposome, since the gas encapsulated liposome with small particle diameter was obtained stably, it discovered that it was useful as an ultrasonic diagnostic agent. Moreover, since this gas-sealed liposome is easily exploded by low frequency ultrasonic irradiation, when a ligand which is bound to a target cell or the like is used as a raw liposome, a gas-sealed liposome having a ligand for the cell or the like is obtained, and the liposome is a target. It can be selectively exploded at the site and found useful as an ultrasonic therapeutic agent or a composition for gene introduction.

That is, the present invention is filled with a fluoride gas or nitrogen gas in an air gap of a sealed container containing a 20 to 80% liposome suspension of an internal volume, and subjected to ultrasonic treatment, and the gas encapsulation thus obtained. It is to provide a liposome.

Moreover, this invention provides the ultrasonic diagnostic agent containing the said gas-sealed liposome.

Moreover, this invention provides the ultrasonic therapeutic agent used by irradiating a low frequency ultrasonic wave to a target site after administration characterized by containing the said gas-sealed liposome.

In addition, the present invention provides a composition for transduction of cells into cells by low-frequency ultrasonic treatment, which comprises the gas-sealed liposomes and genes.

The present invention also provides a method for introducing genes into cultured cells, wherein the gas-sealed liposomes and genes are added to the cultured cells, followed by low frequency ultrasonic irradiation.

The present invention also provides an ultrasonic therapeutic drug to be used by low frequency ultrasonic irradiation of a target site after administration, which comprises the gas-sealed liposome and a drug substance.

Moreover, this invention provides the use for the manufacture of the ultrasonic diagnostic solvent of the said gas encapsulated liposome.

The present invention also provides a use of the gas-sealed liposome for the preparation of an ultrasonic therapeutic drug used by low frequency ultrasonic irradiation of a target site after administration.

Moreover, this invention provides the use for the manufacture of the ultrasonic therapeutic drug used by low frequency ultrasonic irradiation of a target site after administration of the gas-sealed liposome and the composition containing an active ingredient.

Moreover, this invention provides the ultrasonic diagnostic method characterized by administering the said gas encapsulated liposome and then irradiating ultrasonically.

The present invention also provides an ultrasonic therapy comprising the gas encapsulated liposome followed by low frequency ultrasonic irradiation to a target site.

Moreover, this invention provides the ultrasonic therapy characterized by administering the said gas-sealed liposome and a drug substance, and then irradiating a low frequency ultrasonic wave to a target site.

Effects of the Invention

According to the method of the present invention, since the particle diameter depends on the raw material liposomes, gas-sealed liposomes having small and constant particle diameters can be prepared simply and stably. In addition, since it has a small particle diameter, a large amount of gas-sealed liposomes can be transferred to blood clots and atherosclerosis such as arterioles, and imaging of lesions that could not be diagnosed conventionally becomes possible, and blood clots and atherosclerosis of microvascular vessels are also possible. Cattle can be treated.

In addition, the gas-sealed liposome of the present invention has low-frequency ultrasonic irradiation to a target site after administration, thereby generating cavitation by microbubbles of the gas enclosed at the site, and having an explosive action. Therefore, treatment by destruction of a thrombus, an arteriosclerosis, etc. becomes possible. In addition, if the liposome is bound to the ligand for the lesion site, the treatment becomes more site specific. In vivo or in vitro, when gas-sealed liposomes and genes are simultaneously acted on target cells and irradiated with low-frequency ultrasound, microbubbles of gas are generated and cavitation is very effective for target cells. Genetic flow can be introduced.

Implement the invention  Best form for

In the present invention, there is a feature in that liposomes are prepared in advance. The liposome used in the present invention is basically a liposome containing lipids as a membrane constituent, and may have drugs, genes, and the like therein. Repo here.

Lipids used as the membrane constituents of the cotton, cationic lipids in which a primary amino group, secondary amino group, tertiary amino group, or quaternary ammonium group are introduced into these lipids, in addition to phospholipid, glycero glycolipid and sphingolipid glycolipid And lipids having polyalkylene glycol introduced therein as well as lipids bound to ligands for various cells, tissues, and the like.

As phospholipids, for example, phosphatidylcholine (soy phosphatidylcholine, egg yolk phosphatidylcholine, distearoyl phosphatidylcholine, etc.), phosphatidylethanolamine (such as distearoyl phosphatidylethanolamine), phosphatidylserine, phosphatidic acid, phosphatidyl Natural or synthetic phospholipids such as glycerol, phosphatidylinositol, lysophosphatidylcholine, sphingomyelin, egg yolk lecithin, soy lecithin, hydrogenated phospholipids, and the like.

Examples of glycerol glycolipids include sulfoxyribosylglycerides, diglycosyldiglycerides, digalactosyldiglycerides, galactosyldiglycerides, glycosyldiglycerides, and the like. Examples of sphingoglycolipids include galactosyl cerebromide, lactosyl cerebromide, ganglioside and the like.

Examples of cationic lipids include amino groups, alkylamino groups, dialkylamino groups, trialkylammonium groups, monoacyloxyalkyl-dialkylammonium groups, diacyloxyalkyl-monoalkylammonium groups, and the like as the phospholipids, glycerol glycolipids or sphingolipid glycolipids. And lipids into which quaternary ammonium groups are introduced. Further, the polyalkylene glycol in formula lipid, the phospholipid, the glycolipid glycerophosphate, sphingoglycolipids, the formula lipid, polyethylene glycol, polypropylene glycol, such as diethylene -C _{12 - 24} acyl-glycerol-phosphatidylethanolamine Amine-N-PEG etc. are mentioned.

If necessary, cholesterol may be used as a film stabilizer and tocopherols, stearylamine, dicetyl phosphate and ganglioside may be used as an antioxidant.

As ligands for target cells, target tissues, and target lesions, ligands for cancer cells such as transferrin, folic acid, hyaluronic acid, galactose, and mannose; Ligands for thrombus such as RGD peptide, sigma protein, and the like. Monoclonal antibodies and polyclonal antibodies can also be used as ligands.

In addition, the inside of the liposome produced beforehand may be an aqueous phase, and in particular, it does not need to contain a drug substance, genes, etc., but may contain it, when using it as the said therapeutic agent or the composition for gene introduction. As such an active ingredient, For example, Platinum derivatives, such as doxorubicin, 5-FU, Cisplatin, Oxaliplatin, Cancer drug, such as Taxol and a camptothecin; thrombosis therapeutic agents, such as t-PA, urokinase, and stoletokinase; Drugs for treating atherosclerosis such as prostaglandins; Arterial occlusion, NFκ-B for decoction, decoy, and the like. Examples of the genes include DNA, RNA, antisense DNA, siRNA, decoy, therapeutic oligonucleotides, and the like.

Known liposome preparation methods can be applied to the preparation of liposomes, and for example, liposome preparation methods such as Banham [J. Mol. Biol., 13, 238 (1965)], ethanol injection method [J. Cell. Biol., 66, 621 (1975)], French Press Method [FEBS Lett., 99, 210 (1979)], Freeze Thawing Method [Arch. Biochem. Biophys., 212, 186 (1981)], reverse phase evaporation [Proc. Natl. Acad. Sci. USA, 75, 4194 (1978)]. For example, lipids and the like are dissolved in an organic solvent, an aqueous solution is added thereto, followed by sonication to obtain a liposome suspension. This is then established using extruders and / or membrane filters as needed. At this time, the particle diameter is preferably 1 μm or less, more preferably 100 to 800 nm, particularly 100 to 600 nm.

The obtained liposome suspension is injected into a sealed container. At this time, the porosity of the container is preferably 20 to 80%, 30 to 80%, particularly 50 to 80% of the internal volume. If it is less than 20%, the gas introduction rate of the obtained liposome is too low. On the other hand, if it exceeds 80%, it is not economical.

The space is filled with fluoride gas or nitrogen gas. Examples of the fluoride gas include sulfide hexafluoride and perfluoro hydrocarbon gas such as CF ₄ , C ₂ F ₆ , C ₃ F ₈ , C ₄ F ₁₀ , C ₅ F ₁₂ , and C ₆ F ₁₄ . And C ₃ F ₈ , C ₄ F ₁₀ , C ₅ F ₁₂ are particularly preferred. Nitrogen gas can also be used. The pressure after filling is 1 atm (gauge pressure) or more, particularly preferably 1 to 1.5 atm. As a filling means, although it is easy to inject | pour with a syringe etc. from a rubber stopper etc., you may use an injection cylinder.

It is then sonicated. For the ultrasonic treatment, for example, 20 to 50 Hz of ultrasonic waves may be irradiated for 1 to 5 minutes. By the said ultrasonic treatment, the aqueous solution in a liposome, fluoride gas, or nitrogen gas are substituted, and gas-sealed liposome is obtained. The particle diameter of the obtained gas-sealed liposome is almost the same as that of the raw liposome. Therefore, when it establishes at the time of a liposome adjustment, the gas-sealed liposome whose particle diameter is a fixed range, for example, 1 micrometer or less, and 50-800 nm, especially 100-600 nm is obtained easily.

In addition, if a sealed container containing a liposome suspension containing fluoride gas or nitrogen gas is prepared and supplied to a hospital or the like, a gas-sealed liposome can be easily obtained only by ultrasonication in the field.

Since the gas encapsulated liposome thus obtained has a small particle diameter and a uniform particle size distribution, it is possible to move to microvascular vessels, deep tissues and the like. Therefore, the use of gas-sealed liposomes enables not only imaging of microvascular vessels, deep tissues, etc., but also clearer images than conventional ultrasonic diagnostic images using microbubbles. What is necessary is just to perform the ultrasonic diagnosis using the gas-sealed liposome of this invention in accordance with a conventional method. That is, after administering the gas-sealed liposome of this invention, the image of a tissue is obtained by irradiating diagnostic ultrasound (2-6 MHz). As the administration method at this time, intravenous administration etc. are mentioned.

Moreover, when the gas encapsulated liposome of this invention is irradiated with the low frequency ultrasonic wave which contains the resonance frequency of 0.5-2 MHz, liposomal collapse and cavitation by the micro bubble of gas generate | occur | produce. If this cavitation occurs at the thrombus site, the thrombus is destroyed. Thus, if the liposomes have ligands for target lesions, such as thrombus or arteriosclerosis, the gas-sealed liposomes of the present invention administered are bound to thrombus or arteriosclerosis. This appearance can be tracked by diagnostic ultrasound irradiation. When gas-sealed liposomes are bound to a thrombus or atherosclerosis, low-frequency ultrasonic waves can be applied to the site to explode the gas-sealed liposomes, destroy blood clots, and treat them. Examples of diseases that can be treated by the explosion of such gas-sealed liposomes include thrombi, atherosclerosis, vasculitis, and cancer tissues.

In addition, as described above, the gas-sealed liposome of the present invention can contain various medicinal components and genes. Thus, when these drug-containing components or gene-containing liposomes are used, the gas-sealed liposomes can be administered to the target site after administration of the gas-sealed liposomes. When it reaches the target site by the diagnostic ultrasonic wave and irradiates the low frequency ultrasonic wave at the point of reaching the target site, by cavitation generated from the microbubbles of the gas enclosed in the liposome, the active ingredient or gene flow is released at the target site, May be introduced into the target cell. At this time, the drug substance or genes need not be contained in the gas-sealed liposome of the present invention and may be administered at the same time. Here, it is preferable to use liposomes using cationic lipids as liposomes for transgene introduction. In addition, when protamine, polylysine, etc. are also administered simultaneously with genes, the efficiency of gene introduction is further improved.

Herein, the flow rate of the gene flow into the target cell is enhanced by the explosion of the gas-encapsulated liposome. Therefore, after administering the gas-encapsulated liposomes and genes of the present invention, low frequency ultrasound irradiation at the target site enables efficient introduction of genes into target cells. In addition, introduction of the gene into cells can be performed in vitro, that is, cultured cells. In this case, the gas-sealed liposomes and genes of the present invention may be added to the cultured cells, followed by low frequency ultrasonic irradiation.

In addition, in order to efficiently reach the active ingredient and genes of the target cells, target tissues, target lesions, and the like, it is preferable to use liposomes bound to ligands for these target sites as the gas-sealed liposomes of the present invention.

Next, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these at all.

Hereinafter, the abbreviation used by an Example is as follows.

DPPC: dipalmitoylphosphatidylcholine

DPPE: dipalmitoylphosphatidylethanolamine

PEG: polyethylene glycol

Mal: Maltose

DC-Chol: 3β- [N- (N '(N', N'-dimethylaminoethane) carbamoyl] cholesterol

DOPE: Dioleoylphosphatidylethanolamine

DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane

DPTMA: N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride

DDAB: Didodecyldimethylammonium bromide

Example 1

(1) Preparation method of liposome for thrombus targeting

DPPC: Cholesterol: DPPE-PEG: DPPE-PEG-Mal (1: 1: 0.11: 0.02 (m / m)) lipids are dissolved in an organic solvent of chloroform: isopropyl ether (1: 1 v / v), and physiological An aqueous solution (or an aqueous solution containing a drug) such as saline solution was added to a volume of 1/2 of the organic solvent (in this case, chloroform: isopropyl ether: aqueous solution = 1: 1: 1v / v), and mixed to obtain an emulsion. Using this, liposomes were prepared by reverse phase evaporation (REV method). The extruder passes through 400 nm, 200 nm, and 100 nm polycarbonate films to match the particle diameters. By setting it as PEG-liposome, it becomes possible to hold | maintain in dispersion state for a long time (two years). However, at the time of gas encapsulation, since it is encapsulated by ultrasonic waves, since a dispersion state is obtained even by any liposome, the lipid composition is not relevant for gas encapsulation.

CGGGRGDF peptide was added to this liposome solution and allowed to react at room temperature for 1 hour. Subsequently, ultracentrifugation gave RGD-modified PEG-liposomes.

(2) Preparation of cationic liposomes for gene introduction

i) DC-Chol liposome

DOPE: DC-Chol = 2: 3m / m was dissolved in chloroform, placed in a release flask, and the organic solvent was aborated while rotating with a rotary everforate to prepare a thin film of lipid on the wall (preparation of a lipid film). . Liposomes were prepared by hydration with a solvent such as physiological saline. The size is reduced by ultrasonication. Or the particle diameter is made to pass through the polycarbonate film | membrane of 400 nm, 200 nm, and 100 nm with an extruder.

Ii) DOTAP liposomes

DOTAP: DOPE = 1: 1w / w was dissolved in chloroform, placed in a release flask, and rotated with a rotary everphorate to averify an organic solvent to prepare a thin lipid film on the wall (lipid film making). Liposomes were prepared by hydration with a solvent such as physiological saline. The size is reduced by ultrasonication. Or the particle diameter is made to pass through the polycarbonate film | membrane of 400 nm, 200 nm, and 100 nm with an extruder.

The following reagents were used as reagents for gene introduction consisting of commercially available cationic liposomes.

^{Lipofectin TM (DOTMA: DOPE = 1} : 1w / w)

^{LipofectACE TM (DDAB: DOPE = 1} : 1.25w / w)

(3) Method of encapsulating gas into various liposomes

Into a vial bottle (5 ml, 10 ml, 20 ml, etc.), an aqueous liposome solution (lipid concentration: 5 mg / ml) corresponding to 30% (1.5 ml, 3 ml, 6 ml) of the volume was added, and perfluoropropane was added. Substituted with air. Sealed with a rubber stopper, additional perfluoropropane was added by syringe through the rubber stopper. Perfluoro propane was made into a pressurized state of about 1.5 atmospheres by 1.5 times the total volume in the total amount. The bus ultrasonic device 42 ′ was covered with water and allowed to stand, and irradiated for 1 minute.

(4) contrast of blood clots

A balloon catheter is introduced into the iliac artery of the rabbit to stimulate the rubbing of the endothelium to artificially produce a thrombus. When RGD-PEG-liposomes were placed and throttled by the examination ultrasound apparatus (3.5 MHz), the luminance was increased to identify the thrombosis.

(5) rupture of blood clots

A thrombus was produced in the test tube and left for 30 minutes after the addition of the gas-sealed RGD-PEG-liposome obtained in the above (3). The liposome solution was removed, physiological saline was added, and 1 MHz of therapeutic ultrasonic waves were examined. As a result, surface fractures were observed. On the other hand, no crushing was observed when the same operation was performed with gas-sealed PEG-liposomes. This difference was targeted because the binding of the gas-encapsulated liposomes to the thrombi by the RGD peptide occurred.

(6) Human pancreatic cancer AsPC-1 cells (4 × 10 ⁴ cells / well) were cultured in 48 well plates, and FITC-labeled oligonucleotides (18 nucleic acid residues) and gas-encapsulated PEG-liposomes obtained in (3) above were added. Ultrasonic waves of 1 MHz were pulsed for 3 seconds, and the culture solution was immediately washed three to four times. Thereafter, the fluorescence intensity in the cells was observed under a fluorescence microscope. Fluorescence in the cells was observed by low frequency irradiation, and as a result, it was found that the target genes can be introduced into the target cells by adding gas-sealed liposomes and genes to the cells, followed by low frequency irradiation.

(7) Luciferase-coded plasmid DNA and protamine are mixed to prepare a DNA-protamine complex to have a compact size. Incubate AsPC-1 cells (4 × 10 ⁴ cells / well) in the plate of the hole, add DNA-protamine complex (1 μg DNA, lipid: DNA = 12: 1w / w) and gas-sealed PEG-liposome, 1 Ultrasonic waves of MHz were pulsed for 3 seconds, the culture solution was washed three to four times immediately, and the culture solution was added and cultured for two days. Thereafter, luciferase activity was measured by conventional methods. The obtained results are shown in Table 1.

As apparent from Table 1, it was found that the genes can be efficiently introduced into the cells by adding gas-sealed liposomes and genes to the cells, followed by low frequency ultrasonic irradiation.

Claims (15)

A method of producing a gas-sealed liposome, wherein the air gap is filled with a fluoride gas or nitrogen gas in an air gap of a sealed container containing a 20-80% liposome suspension with an inner volume. The method of claim 1, The preparation method wherein the liposome is a cationic lipid containing liposome. The method according to claim 1 or 2, The preparation method wherein the liposome is a liposome having a ligand for a target cell, target tissue or target lesion. Gas-sealed liposome obtained by the manufacturing method of any one of Claims 1-3. Ultrasonic diagnostic solution containing the gas-sealed liposome of Claim 4. The ultrasonic therapeutic agent used by irradiating the low frequency ultrasonic wave to a target site after administration containing the gas-sealed liposome of Claim 4. A gas-incorporated liposome according to claim 4, and a gene-like composition, wherein said composition is introduced into a cell by low-frequency ultrasonic treatment. The gas-sealed liposome according to claim 4 and genes are added to the cultured cells, followed by low frequency ultrasonic irradiation. A method for introducing genes into cultured cells. An ultrasonic therapeutic agent used by low frequency ultrasonic irradiation of a target site after administration, comprising the gas-sealed liposome according to claim 4, and a medicinal component. Use of the gas-sealed liposome according to claim 4 for the preparation of an ultrasonic diagnostic solution. Use for the manufacture of an ultrasonic therapeutic drug, which is used by low frequency ultrasonic irradiation of a target site after administration of the gas-sealed liposome according to claim 4. Use for the manufacture of an ultrasonic therapeutic drug, which is used by low frequency ultrasonic irradiation of a target site after administration of the gas-sealed liposome according to claim 4 and a composition containing the active ingredient. Ultrasonic diagnostic method characterized by administering the gas-sealed liposome according to claim 4, followed by ultrasonic irradiation. Ultrasonic therapy characterized by administering the gas-sealed liposome according to claim 4, followed by low frequency ultrasonic irradiation to the target site. The gas-sealed liposome according to claim 4 and the medicinal component are administered, followed by low-frequency ultrasonic irradiation to a target site.