JP4183047B1 - Self-assembling magnetic lipid nanoparticles usable for gene and drug delivery and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing self-association type magnetic lipid nanoparticles, which can be applied to a drug delivery system and the like, comprising magnetic nanocrystals coated with a fat-soluble surfactant and a fat-soluble drug. In particular, the self-association type simplifies the manufacturing process and facilitates the enhancement of the performance of magnetic lipid nanoparticles by changing the particle composition.
SOLUTION: A lipid-soluble magnetic fluid is prepared by dispersing a magnetic nanocrystal coated with a fat-soluble surfactant in a fat-soluble organic solvent, and a fat-soluble drug or the like is dissolved in the magnetic fluid. Add water-in-oil type reverse micelles by irradiation with ultrasonic waves. Provided is a method for producing self-association-type magnetic lipid nanoparticles, characterized in that a fat-soluble organic solvent is removed by rapid and powerful decompression while irradiating ultrasonic waves following reverse micelles.
[Selection] Figure 1

Description

  The present invention relates to a method for producing self-associating magnetic lipid nanoparticles.

  In the medical field, a drug delivery system aimed at transporting a drug to a lesion site has been developed, but nothing decisive exists yet. In particular, since the elucidation of the function of siRNA (short interfering RNA), the Nobel Prize-winning theme of 2006, siRNA has been highly anticipated as a novel gene therapy drug, but the development of “a method for delivering siRNA to the affected area” Research is very late.

  Under such circumstances, a drug delivery system using magnetic material accumulation by a magnetic field as external energy has been recently developed. One of the main ones of the drug delivery system (Non-patent Document 1) is a magnetic nanocrystal coated with polyethyleneimine (Non-patent Document 2).

  However, nanoparticles using polyethyleneimine as the main raw material are generally limited to (1) in vivo use due to the strong toxicity of polyethyleneimine, and (2) polyethyleneimine itself even in in vitro use. There is a technical problem to be solved that it is not easy to improve the performance of the drug delivery system by changing the structure of the drug. Furthermore, since no clinical trials of gene therapy using nanoparticles whose main ingredient is polyethyleneimine have been reported, that is, there is no past clinical trial data, gene therapy of nanoparticles whose main ingredient is polyethyleneimine Immediate clinical application to is very difficult.

  On the other hand, nanoparticles mainly composed of lipids are (1) it is easy to avoid toxicity by selecting lipids, and (2) there are innumerable lipids that can be selected as nanoparticle materials. By selecting the type, combination, and ratio, it is easy to improve the performance of the drug delivery system (Non-patent Document 3). Furthermore, there have been many clinical trials of gene therapy using nanoparticles mainly composed of lipids.

  Therefore, if magnetic lipid nanoparticles that can be accumulated by a magnetic field can be developed, it can be used as a next-generation drug delivery system that is safe, high-performance, and clinically applicable to a lesion such as a nucleic acid. In particular, development of self-association type magnetic lipid nanoparticles capable of simplifying the production method and the like is desired.

  On the other hand, in the industrial field, a fat-soluble ferrofluid containing magnetic nanocrystals coated with a fat-soluble surfactant (Patent Document 1, Non-Patent Document 4) Since being developed as a seal part for parts, it has been widely used today as a component for various dampers and speakers, a seal part for lubricating hard disk drives, and the like.

Ｒｏｓｅｎｗｅｉｇ， Ｒ．Ｅ． Ｍａｇｎｅｔｉｃ ｆｌｕｉｄｓ．Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｓｃｉｅｎｃｅ ａｎｄ Ｔｅｃｈｎｏｌｏｇｙ ４８−５６（１９６６）． Reimers, G.M. W. & Khalafalla, S .; E. Production of magnetic fluids by peptization techniques. U. S. patent # US3,843,540 (1974). Scherer, F.A. etc. Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo. Gene Therapy 9, 102-9 (2002). Povey, A.M. C. etc. Trapping of chemical carcinogens with magnetic polyethyleneimine microcapsule: microcaps preparation and in vitro reactivity of encapsulated. Journal of Pharmaceutical sciences 75, 831-7 (1986).

Rosenweig, R.W. E. Magnetic fluids. International Science and Technology 48-56 (1966).

  An object of the present invention is to provide a method for producing self-association-type magnetic lipid nanoparticles, which can be applied to a drug delivery system, etc., comprising magnetic nanocrystals coated with a fat-soluble surfactant and a fat-soluble drug. There is to do. In particular, the self-association type simplifies the manufacturing process of magnetic lipid nanoparticles, and facilitates high performance of magnetic lipid nanoparticles by changing the particle composition.

  The inventor has already reported that the performance of the drug delivery system can be improved relatively easily by selecting the type and combination of lipids constituting the lipid nanoparticles (Non-patent Document 3). The present inventor has applied the methodology of fat-soluble ferrofluids (Patent Document 1, Non-Patent Document 4) mainly used in the industrial field, which is a different field, to the technique for producing lipid nanoparticles. The knowledge that can be solved.

  The present invention has been made on the basis of the above knowledge, and (A) a magnetic nanocrystal coated with a fat-soluble surfactant is dispersed in a fat-soluble organic solvent to adjust a fat-soluble magnetic fluid, and the magnetic A step of dissolving a fat-soluble drug or the like in a fluid, adding an aqueous solution or the like, and obtaining a water in oil type reverse micelle in which the magnetic nanocrystals are uniformly dispersed by ultrasonic irradiation or the like; and (B) Subsequently, ultrasonic irradiation or the like is performed to rapidly and powerfully reduce the pressure while maintaining uniform dispersion of the magnetic nanocrystals with high specific gravity. By rapidly evaporating the fat-soluble organic solvent by the reduced pressure, self-association is promoted by a rapid hydrophobic bond between the hydrophobic group of the surfactant and the hydrophobic group of the fat-soluble drug covering the magnetic nanocrystal. Self-association yields self-association-type magnetic lipid nanoparticles with high dispersibility in aqueous solutions by exposing the hydrophilic groups of lipid-soluble drugs to the outermost layer of the nanoparticles through self-association, thereby imparting hydrophilicity to the nanoparticles. Provided is a method for producing type magnetic lipid nanoparticles. By adopting the self-association type, the preparation of the magnetic lipid nanoparticles can be simplified, and the production method can easily improve the performance of the magnetic lipid nanoparticles by changing the particle composition lipid composition and the like.

  The method for producing self-associating magnetic lipid nanoparticles of the present invention comprises (A) preparing a lipid-soluble magnetic fluid by dispersing a magnetic nanocrystal coated with a fat-soluble surfactant in a fat-soluble organic solvent, A step of dissolving a fat-soluble drug in a fluid, adding water or an aqueous solution in which a water-soluble drug is dissolved, and obtaining water-in-oil type reverse micelles in which magnetic nanocrystals are uniformly dispersed by ultrasonic irradiation or the like; And (B) By performing ultrasonic irradiation or the like subsequent to the reverse micelle, rapid and powerful depressurization is performed while maintaining uniform dispersion of the magnetic nanocrystal with high specific gravity. By rapidly evaporating the fat-soluble organic solvent by the reduced pressure, self-association is promoted by a rapid hydrophobic bond between the hydrophobic group of the surfactant and the hydrophobic group of the fat-soluble drug covering the magnetic nanocrystal. By making the hydrophilic group of the lipid-soluble drug appear on the outermost layer of the nanoparticle by the self-association, the nanoparticle is imparted with hydrophilicity to obtain a self-association type magnetic lipid nanoparticle having high dispersibility in an aqueous solution. Features.

  FIG. 1 illustrates the steps of the method for producing self-associating magnetic lipid nanoparticles of the present invention. As shown in FIG. 1, in the method for producing self-associating magnetic lipid nanoparticles of the present invention, (A) a water-in-oil-type reverse micelle is obtained from a mixture of a fat-soluble magnetic fluid in which a fat-soluble drug or the like is dissolved and an aqueous solution. And (B) obtaining self-association type magnetic lipid nanoparticles by reducing the pressure of the reverse micelle under ultrasonic irradiation.

  First, the fat-soluble magnetic fluid used in the present invention will be described. The fat-soluble ferrofluid is composed of a magnetic nanocrystal, a fat-soluble surfactant that coats the magnetic nanocrystal, and a fat-soluble organic solvent. The magnetic nanocrystals coated with the fat-soluble surfactant are uniformly dispersed in the fat-soluble organic solvent through Brownian motion, repulsive force due to the charge of the surfactant, or the like.

Examples of the types of magnetic nanocrystals constituting the fat-soluble magnetic fluid used in the present invention include magnetite (Fe ₃ O ₄ ), maghemite (Fe ₂ O ₃ ), iron monoxide (FeO), and iron (Fe). , Nickel, cobalt, cobalt platinum chromium alloy, barium ferrite alloy, manganese aluminum alloy, iron platinum alloy, iron palladium alloy, cobalt platinum alloy, iron neodymium boron alloy, samarium cobalt alloy, etc. Any of those that can be coated can be used. However, for the purpose of in vivo use, use of magnetite, maghemite, iron monoxide, iron or the like is preferable in order to avoid adverse events due to toxicity. Moreover, it is also possible to mix and use two or more kinds of magnetic nanocrystals as necessary.

  As the kind of the fat-soluble surfactant constituting the fat-soluble magnetic fluid used in the present invention, for example, a fat-soluble unsaturated fatty acid having 18 carbon atoms such as oleic acid, linolenic acid and linolenic acid is preferable. Among them, oleic acid having the smallest number of double bonds, that is, high oxidation resistance is preferable. However, as long as a fat-soluble magnetic fluid can be produced, any type of surfactant can be used without any limitation. Moreover, it is also possible to mix and use 2 or more types of surfactant as needed.

  As a kind of the fat-soluble organic solvent constituting the fat-soluble magnetic fluid used in the present invention, for example, chloroform, n-hexane or the like, which is easily evaporated by reduced pressure with a relatively low boiling point, is coated with a fat-soluble surfactant. Nonpolar fat-soluble organic solvents that can stably disperse magnetic nanocrystals and can dissolve fat-soluble drugs are preferred. However, any fat-soluble organic solvent can be used without any limitation as long as a fat-soluble magnetic fluid capable of dissolving the fat-soluble drug can be produced. Moreover, it is also possible to mix and use 2 or more types of organic solvents as needed.

  As for the fat-soluble drug used in the present invention, for example, phospholipid, glycolipid, sterol, unsaturated or saturated fatty acid, if soluble in a fat-soluble ferrofluid and capable of producing self-associating magnetic lipid nanoparticles, A fat-soluble anticancer agent, a fat-soluble photosensitizer, a fat-soluble contrast agent and the like can be used without any limitation. Moreover, it is also possible to mix and use 2 or more types of fat-soluble chemical | medical agents as needed.

  Examples of the phospholipid used in the present invention include egg yolk lecithin, soybean lecithin, cardiolipin, sphingomyelin, phosphatidic acid, phosphatidylserine, phosphatidylinositol, phosphatidylcholine (for example, dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, Palmitoylphosphatidylcholine, distearoylphosphatidylcholine, etc.), phosphatidylglycerol (eg dioleoylphosphatidylglycerol, dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, etc.), phosphatidylethanolami (E.g., dioleoylphosphatidylethanolamine, dilauroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, etc.) and their hydrogenated products, and the like.

  Examples of the glycolipid used in the present invention include glycosphingolipid (eg, ganglioside, galactosyl cerebroside, lactosyl cerebroside, etc.), glyceroglycolipid (eg, sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride) , Glycosyl diglycerides and the like).

  Examples of sterols used in the present invention include animal-derived sterols (eg, cholesterol, cholesterol succinic acid, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol, etc.), plant-derived sterols (phytosterols) (eg, stigmasterol, sitosterol) , Campesterol, brush casterol, etc.), microorganism-derived sterols (eg, timosterol, ergosterol, etc.) and the like.

  Examples of the saturated or unsaturated fatty acid used in the present invention include unsaturated or saturated fatty acids having 12 to 20 carbon atoms such as palmitic acid, oleic acid, stearic acid, arachidonic acid, and myristic acid.

  Lipids used in the present invention are classified into neutral lipids, positively charged lipids and negatively charged lipids. Examples of neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, cholesterol, ceramide, sphingomyelin, cephalin, and cerebroside. Is mentioned. As positively charged lipids, for example, DOTAP (1,2-dioleoyloxy-3-trimethylammoniumpropane), DC-6-14 (O, O'-diethyldecanoylyl-N- (alpha-trimethylaluminoacetyletyletetylamineetetyl) 3, -N- (N, N, -dimethyl-aminoethane) carbamol cholesterol), TMAG (N- (alpha-trimethylammoniumiocetyl) didodedecyl-D-glutamate chloride), DOTMA (N-2, 3-N-y-pyro, N-y-p-yro, N N-trimethylammon um), DODAC (dioctadecyldimethylammonium chloride), DDAB (didodecylyl-ammonium bromide), DOSPA (2,3-dioyloxylamine-N-dimethyl-N--2). .

  For the purpose of in vivo use, it is preferable to have a hydrophilic polymer on the surface of self-association type magnetic lipid nanoparticles. It is possible to extend the residence time in blood vessels by modifying the hydrophilic polymer on the surface of the self-association type magnetic lipid nanoparticles. Although content of the hydrophilic polymer with respect to the lipid which comprises a self-association type magnetic lipid nanoparticle is not specifically limited, It is 1-10% (molar ratio), Preferably it is 7-10% (molar ratio). The lipid constituting the self-association type magnetic lipid nanoparticle is preferably bonded to the end of the main chain of the hydrophilic polymer, but may be bonded to the side chain.

  The kind of the hydrophilic polymer used in the present invention is not particularly limited. For example, polyalkylene glycol (for example, polyethylene glycol, polypropylene glycol, polytetramethylene glycol, polyhexamethylene glycol), dextran, pullulan, ficoll. , Polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer, amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin, carrageenan, etc., but polyalkylene glycol is preferred. Polyethylene glycol is more preferable.

  When the hydrophilic polymer used in the present invention is a polyalkylene glycol, the molecular weight is usually 300 to 10,000, preferably 1000 to 5,000.

  The hydrophilic polymer used in the present invention includes alkyl groups (for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, s-butyl group, t-butyl group, n- Pentyl group, isopentyl group, t-pentyl group, neopentyl group, etc.), alkoxy group (for example, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, s-butoxy group, t -Butoxy group, etc.), a hydroxyl group, a carbonyl group, an alkoxycarbonyl group, a cyano group and other substituents may be introduced.

  Self-associating magnetic lipid nanoparticle-constituting lipid and hydrophilic polymer are bonded via a covalent bond by reacting the functional group of the self-associating magnetic lipid nanoparticle-constituting lipid with the functional group of the hydrophilic polymer. Can be made. Examples of combinations of functional groups capable of forming a covalent bond include amino group / carboxyl group, amino group / acyl halide group, amino group / N-hydroxysuccinimide ester group, amino group / benzotriazole carbonate group, amino group / Examples include an aldehyde group, a thiol group / maleimide group, and a thiol group / vinylsulfone group.

  Next, the manufacturing method of the self-association type magnetic lipid nanoparticles of the present invention will be described for each step. First, the step (A) will be described. In the step (A), a lipid-soluble magnetic fluid is prepared by dispersing a magnetic nanocrystal coated with a fat-soluble surfactant in a fat-soluble organic solvent, and a fat-soluble drug or the like is dissolved in the magnetic fluid. This is a step of obtaining a water in oil type reverse micelle in which an aqueous solution or the like is added and magnetic nanocrystals are uniformly dispersed by ultrasonic irradiation or the like.

  In the step (A), first, a lipid-soluble magnetic fluid is prepared by dispersing magnetic nanocrystals coated with a fat-soluble surfactant in a fat-soluble organic solvent. As a method for obtaining a fat-soluble magnetic fluid, a conventionally known method can be used without any limitation. Any method may be used as long as the magnetic nanocrystals coated with the fat-soluble surfactant can be dispersed in the fat-soluble organic solvent, but in the present invention, the peptization method is preferably used. By the peptization method, the magnetic nanocrystals are uniformly coated with a fat-soluble surfactant in a short time and dispersed in a fat-soluble organic solvent to form a fat-soluble magnetic fluid.

  The peptization method can be performed by a conventionally known method. Hereinafter, adjustment of the fat-soluble magnetic fluid by the peptization method will be briefly described. The peptization method is divided into a magnetic nanocrystal precipitation step and a magnetic nanocrystal coating step with a fat-soluble surfactant. Hereinafter, a case where magnetite nanocrystals are coated with oleic acid and dispersed in chloroform will be described. The magnetite nanocrystal precipitation step is performed by alkalizing an aqueous solution containing iron ions. The magnetite nanocrystal coating step with such a fat-soluble surfactant is a lipid-soluble that allows uniform dispersion of magnetite nanocrystals in a fat-soluble organic solvent by coating the surface of the obtained magnetite nanocrystal with a fat-soluble surfactant. It is a process performed to adjust the magnetic fluid.

  The magnetite nanocrystal precipitation step in the peptization method is generally used to obtain nano-sized magnetite crystals by alkaline treatment of an aqueous solution containing iron salts such as divalent and trivalent iron chloride, iron sulfate, and iron nitrate. It is a process. The alkali treatment is preferably performed by adding ammonia water. However, as long as magnetite nanocrystals can be precipitated, any type of iron salt or alkali treatment method can be used without any limitation.

  Next, a magnetic nanocrystal coating step with a fat-soluble surfactant in the peptization method will be described. The magnetic nanocrystal coating step with a fat-soluble surfactant in the peptization method involves first adding oleic acid dissolved in kerosene to an ammonia-excess magnetite slurry and stirring well to convert oleic acid into water-soluble ammonium oleate. Replace. By this operation, the hydroxyl group on the surface of the magnetite nanocrystal and the carboxyl group of ammonium oleate are bonded. When heating to 95 degrees Celsius while continuing to stir, ammonium oleate decomposes into fat-soluble oleic acid while generating ammonia gas at 78 degrees Celsius, so the upper kerosene layer and the lower aqueous layer Separate into two phases. This decomposition causes the magnetite nanocrystals to be coated with oleic acid and dispersed in the kerosene layer.

  The kerosene layer in which the oleic acid-coated magnetite nanocrystals are dispersed is taken out and flocculated by adding a polar organic solvent such as acetone, and the flocculation is recovered by permanent magnet or centrifugation. Acetone is added to the collected flocculation, and excess oleic acid is removed by washing the flocculation with a permanent magnet or centrifugation. Subsequently, the oleic acid-coated magnetite nanocrystals are dried by evaporating acetone by reducing the pressure after recovering the flocculation by a permanent magnet or centrifugation.

  By adding chloroform to the obtained oleic acid-coated magnetite nanocrystals and redispersing the oleic acid-coated magnetite nanocrystals in chloroform, a fat-soluble magnetic fluid is obtained.

  In the magnetic nanocrystal coating step with the fat-soluble surfactant in the method for producing self-association type magnetic lipid nanoparticles of the present invention, the magnetic material obtained by the magnetic nanocrystal precipitation step, such as the peptization method described above, is used. By coating the surface of the body nanocrystals with a fat-soluble surfactant, a fat-soluble ferrofluid capable of uniformly dispersing the magnetic nanocrystals in a fat-soluble organic solvent is formed.

  In the step (A), a lipid-soluble magnetic fluid is prepared by dispersing a magnetic nanocrystal coated with a fat-soluble surfactant in a fat-soluble organic solvent, and a fat-soluble drug or the like is dissolved in the magnetic fluid. This is a step of obtaining a water in oil type reverse micelle in which an aqueous solution or the like is added and magnetic nanocrystals are uniformly dispersed by ultrasonic irradiation or the like. Subsequently, water-in in which the magnetic nanocrystals are dispersed uniformly by ultrasonic irradiation or the like by dissolving a fat-soluble drug or the like in the chloroform-based fat-soluble magnetic fluid obtained by the peptization method described above, and further adding an aqueous solution or the like. The process of obtaining oil type reverse micelles will be described. Hereinafter, a process of obtaining water-in-oil type reverse micelles from a mixture obtained by dissolving phospholipids and positively charged lipids in the obtained chloroform-based fat-soluble magnetic fluid and further adding water will be described.

  Phospholipids and positively charged lipids can be obtained by dissolving phospholipids and positively charged lipids in chloroform-based fat-soluble ferrofluids obtained by the peptization method described above, and then irradiating the mixture with water, etc. Water-in-oil type reverse micelles in which water droplets are dispersed in a dissolved chloroform-based fat-soluble ferrofluid are obtained. Note that it is preferable to perform ultrasonic irradiation immediately after the mixture is thoroughly stirred by a vortex method or the like immediately before ultrasonic irradiation. More preferably, the efficiency of stirring can be increased by using glass beads during stirring by the vortex method. This agitation facilitates the formation of water in oil type reverse micelles by ultrasonic irradiation. In addition, since water-in-oil type reverse micelles cause an increase in reverse micelle diameter when left untreated, it is preferable to continue ultrasonic irradiation until immediately before the step (B).

  By the step (A) described above, the lipid-soluble magnetic fluid is prepared by dispersing the magnetic nanocrystals coated with the fat-soluble surfactant in the fat-soluble organic solvent, and the fat-soluble drug or the like is dissolved in the magnetic fluid, Further, an aqueous solution or the like is added, and water in oil type reverse micelles in which the magnetic nanocrystals are uniformly dispersed are formed by ultrasonic irradiation or the like.

  Next, the said process (B) is demonstrated. In the step (B), the reverse micelles obtained in the step (A) are subsequently subjected to ultrasonic irradiation or the like, thereby maintaining a uniform dispersion of magnetic nanocrystals having a high specific gravity in a rapid and powerful manner. Depressurize. By rapidly evaporating the fat-soluble organic solvent by the reduced pressure, self-association is promoted by a rapid hydrophobic bond between the hydrophobic group of the surfactant and the hydrophobic group of the fat-soluble drug covering the magnetic nanocrystal. The self-association causes the hydrophilic group of the fat-soluble drug to be exposed in the outermost layer of the nanoparticle, thereby imparting hydrophilicity to the nanoparticle and forming a self-association type magnetic lipid nanoparticle having high dispersibility in an aqueous solution. Hereinafter, a case where self-association type magnetic lipid nanoparticles are obtained from water in oil type reverse micelles in which water droplets are dispersed in a chloroform-based fat-soluble magnetic fluid in which phospholipids and positively charged lipids are dissolved will be described.

  Continue ultrasonic irradiation or the like on water-in-oil type reverse micelles in which water droplets are dispersed in a chloroform-based lipid-soluble magnetic fluid in which the phospholipids and positively charged lipids obtained in the above step (A) are dissolved After intermittently preventing the reverse micelle diameter from expanding, rapid and powerful pressure reduction is performed using a vacuum pump or the like. In the process of evaporation of the organic solvent due to the reduced pressure, the magnetic nanocrystals are concentrated and the magnetic nanocrystals having a large specific gravity are likely to be separated from the reverse micelles. Is done. Further, in order to effectively prevent the separation, the rapid and strong decompression is preferable. That is, the rapid and powerful decompression forms stable self-associating magnetic lipid nanoparticles by almost completely evaporating the organic solvent before separation of the magnetic nanocrystals from the reverse micelles begins.

  As for the water-soluble drug used in the present invention, buffer salts (for example, phosphate buffer salts, citrate buffer salts, acetate buffer salts, etc.), saccharides, polyhydric alcohols, water-soluble polymers, nonionic surfactants, As long as self-association type magnetic lipid nanoparticles can be produced, such as antioxidants, hydration accelerators, pH adjusters, etc., they can be used without any limitation.

  In the present invention, the ultrasonic irradiation device used in the formation and maintenance of reverse micelles is an open-type supersonic wave in order to prevent oxidation of the raw material of self-associating magnetic lipid nanoparticles and to prevent contamination of constituent metals of the ultrasonic generator. It is preferably a sealed high-power ultrasonic generator such as a cup horn type instead of a sound generator. However, as long as reverse micelles can be formed and maintained, any kind of ultrasonic irradiation device can be used without any limitation.

  By performing ultrasonic irradiation or the like subsequent to the reverse micelle obtained in step (A) by the above-described step (B), rapid and powerful while maintaining uniform dispersion of magnetic nanocrystals with high specific gravity. Depressurize. By rapidly evaporating the fat-soluble organic solvent by the reduced pressure, self-association is promoted by a rapid hydrophobic bond between the hydrophobic group of the surfactant and the hydrophobic group of the fat-soluble drug covering the magnetic nanocrystal. The self-association causes the hydrophilic group of the fat-soluble drug to be exposed in the outermost layer of the nanoparticle, thereby imparting hydrophilicity to the nanoparticle and forming a self-association type magnetic lipid nanoparticle having high dispersibility in an aqueous solution.

  The substance intended for magnetically induced delivery of self-associating magnetic lipid nanoparticles is not particularly limited, and for example, nucleic acids (eg, DNA, RNA, or analogs or derivatives thereof (eg, peptide nucleic acids, Phosphorothioate DNA, etc.), peptides, proteins, drugs (for example, anticancer agents, photosensitizers, contrast agents, etc.), sugars, complexes thereof, etc. The nucleic acids are in single or double stranded form. There is no particular limitation such as linear or annular.

  When the substance to be delivered is a nucleic acid, self-association type magnetic lipid nanoparticle-nucleic acid through electrostatic interaction between negatively charged nucleic acid and positively charged lipid constituting self-association type magnetic lipid nanoparticle A complex is formed, and the complex can be delivered to the target by magnetic induction. However, as long as the target substance can be delivered, the method for binding the target substance to the self-association type magnetic lipid nanoparticles can be used without any particular limitation.

  Self-association type magnetic lipid nanoparticles bound with a target substance or formed a complex with the target substance can be used as a carrier for intracellular delivery of the target substance.

  The biological species from which the target cells to which the target substance is to be delivered is not particularly limited, for example, animals, plants, microorganisms, etc., but is preferably derived from animals, for example, humans, monkeys, cows, sheep More preferred are mammals such as goat, horse, pig, rabbit, dog, cat, mouse, rat and guinea pig. In addition, the type of the target cell is not particularly limited, for example, somatic cells, germ cells, stem cells, or cultured cells thereof.

  Self-association type magnetic lipid nanoparticles bound with a target substance or formed a complex with the target substance can be used both in vivo and in vitro. When used in vivo, administration routes include, for example, intravenous, intraarterial, portal vein, parenchymal organ (eg, brain, eyes, thyroid, mammary gland, heart, lung, liver, pancreas, kidney, adrenal gland, Ovary, testis, etc.), luminal organs (eg, esophagus, stomach, duodenum, jejunum, ileum, large intestine, gallbladder, ureter, intravesical, etc.), cerebrospinal cavity, intrathoracic, intraperitoneal, muscle It is not particularly limited, such as internal, intra-articular, subcutaneous, intradermal.

  In order to improve the uptake efficiency of the self-association type magnetic lipid nanoparticles by the target cells after delivery of the self-association type magnetic lipid nanoparticles bound to the target substance or formed a complex with the target substance to the target cells If necessary, a substance capable of binding to a receptor on the cell membrane surface (for example, an antibody or a fragment thereof (for example, Fab fragment, F (ab) ′ 2 fragment, single chain antibody, etc.), insulin, transferrin, folic acid, hyaluron Acid, sugar chain, apolipoprotein (eg, apo A-1, apo B-48, apo B-100, apo E, etc.), growth factor (eg, epidermal growth factor, hepatocyte growth factor, fibroblast growth factor, Insulin-like growth factor, etc.) and the like can be bound to the surface of the self-association type magnetic lipid nanoparticles without any particular limitation.

References

JP 49-84998
Japanese Patent Publication 2006-167521

The invention's effect

  When self-associating magnetic lipid nanoparticles are produced using the method for producing self-associating magnetic lipid nanoparticles of the present invention, lipid nanoparticles can be accumulated by a magnetic field, so that genes, anticancer agents, photosensitivity It can be used as a delivery system for various drugs such as substances. Compared with the polyethyleneimine-coated magnetic nanocrystals of the prior art, the self-association type magnetic lipid nanoparticles of the present invention can easily improve the performance of nanoparticles and avoid toxicity by changing the composition. High design freedom.

  Hereinafter, the present invention will be described in more detail with reference to examples. Needless to say, the scope of the present invention is not limited to such examples.

Example 1 (Process A)
0.04 mol of iron (I) chloride tetrahydrate and 0.08 mol of iron (II) chloride hexahydrate were dissolved in 50 ml of deionized water. While stirring the obtained aqueous iron chloride solution at room temperature with vigorous stirring using a stirrer bar and a temperature-adjustable stirrer, 50 ml of aqueous ammonia was added at a rate of 1 to 2 ml per second to obtain a slurry of magnetite nanocrystals. . Further, while stirring with a stirrer bar, the mixture was heated to 95 degrees Celsius and 55 ml of kerosene containing 10% by mass of oleic acid was added at a rate of 2 to 3 ml per second. In this process, the fat-soluble oleic acid is replaced with water-soluble ammonium oleate, and the hydroxyl group on the surface of the magnetite nanocrystal and the carboxyl group of ammonium oleate are bonded. Furthermore, since the produced ammonium oleate is decomposed into oleic acid while generating ammonia gas at 78 degrees Celsius, the magnetite nanocrystals are coated with fat-soluble oleic acid and uniformly dispersed in the upper kerosene layer. . After confirming uniform dispersion, most of the lower aqueous layer containing a large amount of ammonium chloride was removed with a Pasteur pipette, and heating was continued at 95 degrees Celsius until the aqueous layer was completely evaporated. 10 ml of the obtained kerosene layer was placed in a 200 ml beaker, and 100 ml of acetone was added to flocculate the oleic acid-coated magnetite nanocrystals. The flocculation was magnetically accumulated by applying a 1.4 Tesla permanent magnet from the outside of the beaker and leaving it at room temperature for 5 minutes. The supernatant was discarded by decantation while the magnet was placed outside the beaker and the flocculation was accumulated. After adding 80 ml of acetone to the obtained flocculation and stirring gently for 1 to 2 minutes, the supernatant is decanted again using a magnet in the same manner, so that excess olein is removed from the oleic acid-coated magnetite nanocrystals. The acid was removed. Furthermore, acetone was completely removed from the oleic acid-coated magnetite nanocrystals by performing a vacuum treatment at 25 degrees Celsius and 50 mmHg for 8 hours using a vacuum oven. Analysis by gas chromatography revealed that the oleic acid-coated magnetite nanocrystals contained 19.2% by weight of oleic acid. By redispersing 10 mg of the obtained oleic acid-coated magnetite nanocrystals in 10 ml of chloroform, a fat-soluble magnetic fluid was formed.

  0.4 ml of the obtained magnetic fluid was placed in a 20 ml pear-shaped flask, and dioleoylphosphatidylethanolamine (Dioleoylphosphatidylethanolamine, hereinafter referred to as “DOPE”, purchased from SIGMA) 0.516 mg and N- [1- (2,3-dioleoyloxy) propyl) -N, N, N-trimethylammonium chloride (N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride, hereinafter “DOTAP” Add 0.484 mg (purchased from SIGMA) and stir well. Add 0.6 ml of chloroform to the resulting mixture and adjust the whole volume to 1 ml. Furthermore, 1 ml of distilled water and 30 1 mm diameter glass beads are added, the inside of the pear flask is replaced with argon gas to prevent oxidation, sealed with a glass stopper, and then heated to 37 degrees Celsius using a hot tub. . After the heating, the mixture is stirred for 1 minute at the maximum frequency using a vortex mixer, the glass stopper is removed, and immediately after that, it is connected to a rotary evaporator. At the same time that the rotational speed of the rotary evaporator is maximized, the pear-shaped flask portion is put into a cup of a cup horn type sonicator, and ultrasonic irradiation is performed at the maximum output for 1 minute. In addition, 37 degrees Celsius hot water is circulated in advance in the cup portion. By the ultrasonic irradiation, reverse micelles are formed in which small water droplets of distilled water are dispersed in a fat-soluble magnetic fluid composed of oleic acid-coated magnetite nanocrystals, DOPE, DOTAP and chloroform.

(Process B)
After the ultrasonic irradiation, the pressure is rapidly reduced at a maximum speed to 50 mmHg using a vacuum pump connected to a rotary evaporator while continuing the ultrasonic irradiation at the maximum output. Due to this reduced pressure, chloroform is evaporated from the reverse micelles, and the hydrophobic group of oleic acid, the hydrophobic group of DOPE, and the hydrophobic group of DOTAP that coat the magnetite nanocrystals are bonded together by a hydrophobic bond. By this binding, DOPE and DOTAP coat the surface of the oleic acid-coated magnetite nanocrystal with the hydrophilic group directed to the outermost layer of the nanoparticle, thereby forming a self-association type magnetic lipid nanoparticle that is stably dispersed in distilled water. . In addition, in the process of evaporation of chloroform mentioned above, since it involves scattering of reverse micelles inside the pear-shaped flask, after returning to normal pressure by replacing the inside of the flask with an inert gas such as argon gas as necessary, The flask is removed from the rotary evaporator, sealed with a glass stopper, and the reverse micelles scattered by centrifugation using a rotary centrifuge are collected at the bottom of the flask.

  In order to remove chloroform from the obtained self-association type magnetic lipid nanoparticles, a rotary evaporator is used and a vacuum treatment of 10 mmHg is performed for 15 minutes in a 20 ° C. hot tub. Since the distilled water evaporates due to the reduced pressure, the flask is removed from the rotary evaporator after returning to normal pressure by replacing the inside of the flask with an inert gas such as argon gas as necessary. Add the appropriate distilled water and stir well. Distilled water is added to the obtained self-association type magnetic lipid nanoparticle dispersion water to a total volume of 1.4 ml and stirred well to form self-association type magnetic lipid nanoparticles having a concentration of 1 mg / ml. FIG. 2 shows a transmission electron microscope (JEM1200EX, JEOL) photograph after negative staining of the obtained self-association type magnetic lipid nanoparticles. As shown in FIG. 2, the magnetic lipid nanoparticles obtained according to the present invention were self-associating and had a structure in which the magnetite nanocrystals were coated with lipid. Moreover, in the particle diameter measurement by a light-scattering spectrometer (ELS-8000, Otsuka Electronics), the average diameter of the obtained self-association type magnetic lipid nanoparticle was 358.2 nm, which was consistent with the result of the electron micrograph. From the above results, it was confirmed that magnetic lipid nanoparticles were successfully formed by self-association.

Example 2
As a fat-soluble drug added to the obtained magnetic fluid, O, O′-ditetradecanoyl-N- (alpha-trimethylammonioacetyl) -diethanolamine chloride (O, O′-ditetradecanoyl-N- (alpha-trimethyl-) Self-association type magnetic lipid nanoparticles were obtained in the same manner as in Example 1 except that 0.690 mg (provided by ammonioacetyl) diethanolamine chloride (Daiichi Pharmaceutical Co., Ltd.) and 0.310 mg of DOPE were used. The obtained self-association type magnetic lipid nanoparticles also had the same shape as that obtained in Example 1 in the analysis with an electron microscope, but had a smaller nanoparticle diameter (FIG. 3). ). Moreover, in the measurement by the light scattering spectrometer, the average diameter of the obtained self-association type magnetic lipid nanoparticles was 165.8 nm, which was consistent with the result of the electron micrograph. From the above results, it was confirmed that magnetic lipid nanoparticles were successfully formed by self-association.

Example 3
In this example, Allstars Transfection Control siRNA (purchased from Qiagen, hereinafter referred to as “siRNA ^Control ”), which is a siRNA having a gene sequence that has been confirmed not to inhibit all messenger RNA (mRNA) in human cells, is negative. Allstars, which is a siRNA having a gene sequence that has been confirmed to inhibit mRNA of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which is a house key pink gene, as a control Transfection ^GAPDH siRNA (purchased from Qiagen, hereinafter referred to as “siRNA ^GAPDH ”) Used as

A human gastric cancer cell line KATOIII (1.0 × 10 ⁴ cells / 100 μl) suspended in RPMI 1640 medium without serum and antibiotics was cultured on a 96-well cell culture plate for 12 hours. RPMI 1640 medium was added to 0.375 μl of the self-association type magnetic lipid nanoparticle dispersion obtained in Example 2 to a total of 25 μl and incubated at room temperature for 5 minutes. To the obtained self-association type magnetic lipid nanoparticle / RPMI1640 dispersion, 25 μl of siRNA (siRNA ^Control or siRNA ^GAPDH ) at a concentration of 300 nM was added and incubated at room temperature for 10 minutes. After removing the KATOIII culture solution on the 96-well cell culture plate, the obtained 50 μl self-association type magnetic lipid nanoparticle / siRNA complex was added.

  A cell is irradiated with a magnetic field by press-fitting a cylindrical 0.2 Tesla neodymium magnet having a diameter of 6 mm and a length of 8 mm to the well portion of the bottom of the culture plate to which the complex is added, and the cells are cultured at 37 degrees for 10 minutes. Performed (magnetic field irradiation group). Alternatively, the culture was performed at 37 ° C. for 10 minutes without applying a magnetic field (magnetic field non-irradiation group). Subsequently, after removing the complex, 200 μl of RPMI1640 medium containing no serum and antibiotics was added, and culturing was performed for 48 hours.

  The culture solution was removed, and the cells were further washed twice with 250 μl of phosphate buffered saline, followed by GAPDH enzyme quantification after cell lysis treatment. Cell lysis treatment and GAPDH enzyme quantification were performed using GAPDH enzyme quantification cell lysate (purchased from Ambion), GAPDH enzyme quantification reagent kit (KDalert reagent kit, purchased from Ambion), fluorescence absorptiometer (Perkin Elmer) And according to the manual of the kit.

When the GAPDH enzyme activity was quantified as described above, the “siRNA ^GAPDH + non-magnetic field irradiation” group showed 96.1% GAPDH enzyme activity of the “siRNA ^Control + magnetic field irradiation” group (ie, negative control group) (ie, The GAPDH enzyme inhibitory effect under non-magnetic field irradiation by the self-associating magnetic lipid nanoparticles was only 3.9%). On the other hand, the “siRNA ^GAPDH + magnetic field irradiation” group showed 43.6% GAPDH enzyme activity of the “siRNA ( ^Control + magnetic field irradiation) group (ie, negative control group)” (ie, self-associated magnetic lipid nanoparticles). The GAPDH enzyme inhibitory effect under magnetic field irradiation was 56.4%, and the inhibitory effect was 14 times or more under non-magnetic field irradiation.) From the above results, the self-association type magnetic lipid nanoparticle was It was confirmed that it was useful as a magnetic induction carrier.

  As described above, the self-association type magnetic lipid nanoparticles of the present invention can be used in the biotechnology field such as gene transfer into cells and animal tissues by changing the type and mixing ratio of the fat-soluble drug to be constituted. In addition to treating diseases by delivering genes and drugs to the affected area, detection of self-assembled magnetic lipid nanoparticles accumulated in the affected area with a micro magnetic detection device. It can also be used for diagnosis of diseases by detecting self-associating magnetic lipid nanoparticles.

  2 illustrates the steps of the method for producing self-associating magnetic lipid nanoparticles of the present invention. In 1), since the specific gravity of the fat-soluble magnetic fluid containing the fat-soluble drug exceeded the specific gravity of the aqueous solution containing the water-soluble drug, the aqueous solution having a small specific gravity is the upper layer, and the fat-soluble magnetic fluid having the high specific gravity is the lower layer. The state of forming is illustrated. If the specific gravity of the aqueous solution exceeds the specific gravity of the fat-soluble magnetic fluid, the fat-soluble magnetic fluid forms the upper layer and the aqueous solution having a higher specific gravity forms the lower layer.   It is the transmission electron micrograph after negative dyeing | staining of the self-association type magnetic lipid nanoparticle obtained in Example 1 by the manufacturing method of the self-association type magnetic lipid nanoparticle of this invention.   It is the transmission electron micrograph after negative dyeing | staining of the self-association type magnetic lipid nanoparticle obtained in Example 2 by the manufacturing method of the self-association type magnetic lipid nanoparticle of this invention.

Claims (5)

(A) A lipid-soluble magnetic fluid is prepared by dispersing magnetic nanocrystals coated with a fat-soluble surfactant in a fat-soluble organic solvent, the fat-soluble drug is dissolved in the magnetic fluid, and an aqueous solution is further added. A step of obtaining water-in-oil type reverse micelles in which magnetic nanocrystals are uniformly dispersed by ultrasonic irradiation; and (B) uniform irradiation of magnetic nanocrystals having a high specific gravity by performing ultrasonic irradiation following the reverse micelles. In this state, a rapid and powerful pressure reduction is performed, the lipophilic organic solvent is rapidly evaporated by the pressure reduction, and the hydrophobic group of the surfactant and the fat-soluble drug that coat the magnetic nanocrystals by the evaporation. of encourage self-association by rapid hydrophobic bond between a hydrophobic group, a hydrophilic group of the lipophilic drug by exposed in the outermost layer of the nanoparticles by the self-association, hydrophilicity imparted to the nanoparticles, water Method for producing a self-associated magnetic lipid nanoparticles to obtain high self-associated magnetic lipid nanoparticles dispersibility in a liquid. The method for producing self-associating magnetic lipid nanoparticles according to claim 1, wherein self-associating magnetic lipid nanoparticles are obtained by changing the amount and ratio of the fat-soluble magnetic fluid and the fat-soluble drug, or the reaction temperature . The self-association type magnetic lipid according to any one of claims 1 to 2, wherein the ultrasonic irradiation is performed continuously or intermittently for the purpose of maintaining uniform dispersion of magnetic nanocrystals having high specific gravity. A method for producing nanoparticles. The method for producing self-association type magnetic lipid nanoparticles according to any one of claims 1 to 3, wherein the reduced pressure promotes self-association of the magnetic nanoparticles by changing the reduced pressure strength and the reduced pressure rate .   Self-associated magnetic lipid nanoparticles obtained by the method for producing self-associated magnetic lipid nanoparticles according to any one of claims 1 to 4.

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