JP5904555B2 - Method for producing liposome - Google Patents
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- JP5904555B2 JP5904555B2 JP2012553652A JP2012553652A JP5904555B2 JP 5904555 B2 JP5904555 B2 JP 5904555B2 JP 2012553652 A JP2012553652 A JP 2012553652A JP 2012553652 A JP2012553652 A JP 2012553652A JP 5904555 B2 JP5904555 B2 JP 5904555B2
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- microchannel
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- 239000002502 liposome Substances 0.000 title claims description 150
- 238000004519 manufacturing process Methods 0.000 title claims description 85
- 150000003904 phospholipids Chemical class 0.000 claims description 86
- 239000006185 dispersion Substances 0.000 claims description 55
- 239000003960 organic solvent Substances 0.000 claims description 40
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 28
- 239000008393 encapsulating agent Substances 0.000 claims description 16
- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 14
- 239000001569 carbon dioxide Substances 0.000 claims description 14
- 239000003795 chemical substances by application Substances 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 11
- 238000011085 pressure filtration Methods 0.000 claims description 9
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 239000007789 gas Substances 0.000 description 42
- 238000000034 method Methods 0.000 description 29
- 239000002245 particle Substances 0.000 description 28
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- FQZQXPXKJFOAGE-KICCZPNWSA-N [(2r)-3-[hydroxy-[(5r)-2,3,4,5,6-pentahydroxycyclohexyl]oxyphosphoryl]oxy-2-octadecanoyloxypropyl] octadecanoate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@@H](OC(=O)CCCCCCCCCCCCCCCCC)COP(O)(=O)OC1C(O)C(O)C(O)[C@@H](O)C1O FQZQXPXKJFOAGE-KICCZPNWSA-N 0.000 description 2
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- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
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- JQWAHKMIYCERGA-UHFFFAOYSA-N (2-nonanoyloxy-3-octadeca-9,12-dienoyloxypropoxy)-[2-(trimethylazaniumyl)ethyl]phosphinate Chemical compound CCCCCCCCC(=O)OC(COP([O-])(=O)CC[N+](C)(C)C)COC(=O)CCCCCCCC=CCC=CCCCCC JQWAHKMIYCERGA-UHFFFAOYSA-N 0.000 description 1
- ASWBNKHCZGQVJV-UHFFFAOYSA-N (3-hexadecanoyloxy-2-hydroxypropyl) 2-(trimethylazaniumyl)ethyl phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(O)COP([O-])(=O)OCC[N+](C)(C)C ASWBNKHCZGQVJV-UHFFFAOYSA-N 0.000 description 1
- WRGQSWVCFNIUNZ-GDCKJWNLSA-N 1-oleoyl-sn-glycerol 3-phosphate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)COP(O)(O)=O WRGQSWVCFNIUNZ-GDCKJWNLSA-N 0.000 description 1
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 1
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000004909 Moisturizer Substances 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- ATBOMIWRCZXYSZ-XZBBILGWSA-N [1-[2,3-dihydroxypropoxy(hydroxy)phosphoryl]oxy-3-hexadecanoyloxypropan-2-yl] (9e,12e)-octadeca-9,12-dienoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCC\C=C\C\C=C\CCCCC ATBOMIWRCZXYSZ-XZBBILGWSA-N 0.000 description 1
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- 230000003712 anti-aging effect Effects 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
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- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 239000007844 bleaching agent Substances 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 150000001840 cholesterol esters Chemical class 0.000 description 1
- 235000019416 cholic acid Nutrition 0.000 description 1
- 239000002812 cholic acid derivative Substances 0.000 description 1
- 150000001842 cholic acids Chemical class 0.000 description 1
- 239000002872 contrast media Substances 0.000 description 1
- RNPXCFINMKSQPQ-UHFFFAOYSA-N dicetyl hydrogen phosphate Chemical compound CCCCCCCCCCCCCCCCOP(O)(=O)OCCCCCCCCCCCCCCCC RNPXCFINMKSQPQ-UHFFFAOYSA-N 0.000 description 1
- 229940093541 dicetylphosphate Drugs 0.000 description 1
- 235000013681 dietary sucrose Nutrition 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000002296 dynamic light scattering Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 235000019410 glycyrrhizin Nutrition 0.000 description 1
- 239000007952 growth promoter Substances 0.000 description 1
- 230000003779 hair growth Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- BHEPBYXIRTUNPN-UHFFFAOYSA-N hydridophosphorus(.) (triplet) Chemical compound [PH] BHEPBYXIRTUNPN-UHFFFAOYSA-N 0.000 description 1
- 238000009652 hydrodynamic focusing Methods 0.000 description 1
- 150000004679 hydroxides Chemical class 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001333 moisturizer Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 1
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 1
- 150000003905 phosphatidylinositols Chemical class 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 229920000233 poly(alkylene oxides) Polymers 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 229960004793 sucrose Drugs 0.000 description 1
- 150000005846 sugar alcohols Polymers 0.000 description 1
- -1 that is Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 239000002691 unilamellar liposome Substances 0.000 description 1
- 239000004034 viscosity adjusting agent Substances 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1277—Processes for preparing; Proliposomes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
- B01J13/04—Making microcapsules or microballoons by physical processes, e.g. drying, spraying
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Dispersion Chemistry (AREA)
- Organic Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Preparation (AREA)
- Manufacturing Of Micro-Capsules (AREA)
Description
本発明は、有機溶媒を使用することなく、さらに炭酸ガス等による加圧濾過や、加熱を行うことなく、粒子径が均一化されたリポソームを容易に製造しうるリポソームの製造方法に関し、及び該製造方法により得られたリポソームに関する。さらには、このようなリポソーム製造用のデバイス並びに製造用キットに関する。 The present invention relates to a method for producing a liposome capable of easily producing a liposome having a uniform particle size without using an organic solvent, further by pressure filtration with carbon dioxide gas or the like, or without heating, and The present invention relates to a liposome obtained by the production method. Furthermore, the present invention relates to such a device for producing liposomes and a production kit.
本出願は、参照によりここに援用されるところの日本出願特願２０１１−１０４０８号優先権を請求する。 This application claims Japanese application Japanese Patent Application No. 2011-10408 priority used here by reference.
リポソームは、主にリン脂質によって形成される二分子膜（リポソーム膜）の閉鎖小胞体であり、生体膜と類似の構造や機能を有するため、従来から様々な研究材料として用いられてきている。このリポソームは、内部に有する水相には、水溶性の内包剤（例えば、薬効成分）を、二分子膜の内部には油溶性の内包剤を保持するという、いわゆるカプセル構造を構築できることから、診断、治療、化粧などの様々な分野で用いられてきている。さらに、近年では、薬物送達システム（ＤＤＳ）への応用が盛んに研究されている。 Liposomes are closed vesicles of bilayer membranes (liposome membranes) formed mainly by phospholipids and have similar structures and functions as biological membranes, and thus have been used as various research materials. Since this liposome can construct a so-called capsule structure in which a water-soluble inclusion agent (for example, a medicinal ingredient) is retained in the aqueous phase inside and an oil-soluble inclusion agent is retained inside the bilayer membrane, It has been used in various fields such as diagnosis, treatment, and makeup. Furthermore, in recent years, application to drug delivery systems (DDS) has been actively studied.
このような薬効成分などの内包剤を内包したリポソームは、ロータリエバポレータ等を使い、恒温槽にて丸底フラスコの回転条件下でフラスコの底部に脂質膜を形成させ、そこに内包物を含む水溶液を導入して、減圧条件下で形成されるバンガム（Bangham）法がある（非特許文献１）。かかるバンガム法により得られるリポソームは粒子径分布が広く、ゲルろ過等の処理を経て所望の粒径のリポソームの回収を行っていた。しかし、その方法では回収されるリポソームは投入原料のせいぜい１％程度にしかならず、製造コストの高額化が大きな問題であった。 Liposomes encapsulating such encapsulating agents such as medicinal components are aqueous solutions that use a rotary evaporator or the like to form a lipid film at the bottom of the flask under the rotating condition of a round bottom flask in a thermostatic bath, and contain the inclusions therein. There is a Bangham method that is formed under reduced pressure conditions (Non-patent Document 1). Liposomes obtained by such a bangham method have a wide particle size distribution, and liposomes having a desired particle size have been collected through treatment such as gel filtration. However, in this method, the recovered liposome is only about 1% of the input raw material, and an increase in production cost has been a big problem.
マイクロ流路を使ったリポソームの製法としてはアンドレアス・ジャーン等の方法がある（非特許文献２）。この方法では、流路が十字構造となり、中央流路に内包物と脂質と有機溶媒の混合物を流し、両側流路から緩衝液を流し、この両者の混合速度（希釈過程）の調整によりリポソームを製造する。また、他の製造方法として、有機溶媒に溶解させたリン脂質溶液をマイクロ流路に導入し、有機溶媒を乾燥させてマイクロ流路の壁にリン脂質の薄膜を形成させ、リポソームを生成させる方法について開示がある（特許文献１）。上記の方法では、有機溶媒を除去する工程が煩雑であり、また有機溶媒がリポソーム製剤に残留する可能性は完全には否定することができず、有機溶媒による生体への好ましくない影響が問題となる。係るマイクロ流路を用いる方法によれば、粒子径が均一化されたリポソームを容易に製造することができるが、有機溶媒を除去したり、乾燥させる工程に、時間を要することが問題であった。 As a method for producing a liposome using a microchannel, there is a method such as Andreas Jahn (Non-patent Document 2). In this method, the flow path has a cross-shaped structure, and a mixture of inclusions, lipids, and an organic solvent is allowed to flow through the central flow path, a buffer solution is flowed from both flow paths, and liposomes are adjusted by adjusting the mixing speed (dilution process) of both. To manufacture. As another production method, a phospholipid solution dissolved in an organic solvent is introduced into a microchannel, and the organic solvent is dried to form a phospholipid thin film on the wall of the microchannel to generate liposomes. Is disclosed (Patent Document 1). In the above method, the process of removing the organic solvent is complicated, and the possibility that the organic solvent remains in the liposome preparation cannot be completely ruled out. Become. According to the method using such a microchannel, liposomes having a uniform particle diameter can be easily produced, but it has been a problem that it takes time to remove the organic solvent or to dry it. .
有機溶媒を用いないで、リポソームを生成させる方法として、リポソーム膜成分物質と水溶液とをリポソーム膜成分物質の相転移温度以上で混合し、炭酸ガスによる加圧濾過を行う方法（特許文献２）、ヒドロキシル基を有する化合物の存在下で、脂質膜成分と超臨界若しくは亜臨界状態の二酸化炭素（炭酸ガス）とを混合することにより作製する方法（特許文献３）などが開示されている。しかしながら、これらの方法は、炭酸ガスによる加圧濾過や、又は超臨界若しくは亜臨界状態の二酸化炭素（炭酸ガス）を要し、煩雑な工程を要する。また、有機溶媒を含まないリポソーム含有製剤について開示があるが、係るリポソーム含有製剤の製造工程においても、リポソーム膜成分物質の４０〜６５℃の相転移温度でリポソーム膜成分物質を処理し、二酸化炭素（炭酸ガス）を用いることも要件とされている（特許文献４）。 As a method of generating liposomes without using an organic solvent, a method of mixing a liposome membrane component substance and an aqueous solution at a phase transition temperature or higher of the liposome membrane component substance and performing pressure filtration with carbon dioxide gas (Patent Document 2), A method of producing a lipid membrane component by mixing a supercritical or subcritical carbon dioxide (carbon dioxide gas) in the presence of a compound having a hydroxyl group (Patent Document 3) is disclosed. However, these methods require pressure filtration with carbon dioxide gas, or supercritical or subcritical carbon dioxide (carbon dioxide gas), which requires complicated steps. Moreover, although there is a disclosure of a liposome-containing preparation that does not contain an organic solvent, in the production process of the liposome-containing preparation, the liposome membrane component substance is treated at a phase transition temperature of 40 to 65 ° C. The use of (carbon dioxide) is also a requirement (Patent Document 4).
本発明は、有機溶媒を使用することなく、さらに炭酸ガス等による加圧濾過や、有機溶媒を除去するための加熱処理、吸引処理や乾燥処理を行うことなく、粒子径が均一化されたリポソームを容易に製造しうるリポソームの製造方法を提供することを課題とする。また、そのような該製造方法により得られたリポソームを提供することを課題とし、さらには、このようなリポソーム製造用のデバイス並びに製造用キットを提供することを課題とする。 The present invention provides a liposome having a uniform particle size without using an organic solvent, and without performing pressure filtration with carbon dioxide gas, etc., heat treatment for removing the organic solvent, suction treatment or drying treatment. It is an object of the present invention to provide a method for producing liposomes that can be easily produced. It is another object of the present invention to provide a liposome obtained by such a production method, and further to provide a device and a production kit for producing such a liposome.
本発明者らは、上記課題を解決するために鋭意検討を重ねた結果、リポソームの生成工程において、マイクロ流路に、リン脂質分散液、及び気体、即ち空気、不活性ガス、若しくはそれらの混合物を主成分とする気体を導入し、当該マイクロ流路内でリン脂質分散液と気体を混合させる工程を含むことを特徴とする製造方法によれば、有機溶媒を使用することなく、さらに炭酸ガス等による加圧濾過や、有機溶媒を除去するための加熱処理、吸引処理や乾燥処理を行うことなく、粒子径が均一化されたリポソームを製造しうることを見出し、本発明を完成した。 As a result of intensive studies in order to solve the above-mentioned problems, the present inventors have found that in the liposome production process, the phospholipid dispersion liquid and gas, that is, air, inert gas, or a mixture thereof are provided in the microchannel. According to the manufacturing method, which includes the step of introducing a gas mainly containing a gas and mixing the phospholipid dispersion and the gas in the microchannel, the carbon dioxide gas is further used without using an organic solvent. The inventors have found that liposomes having a uniform particle diameter can be produced without performing pressure filtration by means of, etc., heat treatment for removing the organic solvent, suction treatment or drying treatment, thereby completing the present invention.
つまり、本発明は以下からなる。
１．リポソームの生成工程において、マイクロ流路に、有機溶媒を含まない溶液とリン脂質成分からなるリン脂質分散液、及び気体を導入し、当該マイクロ流路内でリン脂質分散液と気体を混合させる工程を含むことを特徴とするリポソームの製造方法。
２．前記リポソームの生成工程において、炭酸ガスによる加圧濾過工程を含まないことを特徴とする、前項１に記載のリポソームの製造方法。
３．マイクロ流路が、導入部に少なくとも２つのマイクロ流路を有し、導出部に１つのマイクロ流路を有する分岐型マイクロ流路であって、以下の工程を含むことを特徴とする、前項１又は２に記載のリポソームの製造方法：
１）導入部における１のマイクロ流路よりリン脂質分散液を導入する工程；
２）導入部における他の１のマイクロ流路より気体を導入する工程；
３）マイクロ流路内で、リン脂質分散液と気体を混合させる工程；
４）導出部のマイクロ流路よりリポソームを導出する工程。
４．マイクロ流路の内径が、１００〜１０００μｍである、前項１〜３のいずれか１に記載のリポソームの製造方法。
５．リン脂質分散液流量：気体流量が、０．００１〜１０：１である、前項１〜４のいずれか１に記載のリポソームの製造方法。
６．気体流量が、１〜１５ｍｌ／時である、前項１〜５のいずれか１に記載のリポソームの製造方法。
７．リポソーム形成時の温度が、１５〜８０℃である、前項１〜６のいずれか１に記載のリポソームの製造方法。
８．前項１〜７のいずれか１に記載のリポソームの製造方法において、マイクロ流路に、さらに内包剤を導入し、当該マイクロ流路内でリン脂質分散液、内包剤及び気体を混合することを特徴とする、内包剤を含有するリポソームの製造方法。
９．前項１〜８のいずれか１に記載のリポソームの製造方法により得られるリポソーム。
１０．マイクロ流路を含んでなる、前項９に記載のリポソームの製造用デバイス。
１１．前項１０に記載のリポソーム製造用デバイスと、有機溶媒を含まないリン脂質分散液、内包剤及び／又は気体の導入用シリンジを含むことを特徴とする、リポソーム製造用キット。That is, this invention consists of the following.
1. In the liposome production step, a step of introducing a solution containing no organic solvent and a phospholipid dispersion composed of a phospholipid component and a gas into the microchannel, and mixing the phospholipid dispersion and the gas in the microchannel A method for producing a liposome, comprising:
2. 2. The method for producing a liposome according to item 1 above, wherein the liposome production step does not include a pressure filtration step using carbon dioxide gas.
3. Item 1. The microchannel is a branched microchannel having at least two microchannels in the introduction part and one microchannel in the lead-out part, and includes the following steps: Or the manufacturing method of the liposome as described in 2:
1) a step of introducing a phospholipid dispersion from one microchannel in the introduction part;
2) a step of introducing a gas from the other one microchannel in the introduction section;
3) mixing the phospholipid dispersion and gas in the microchannel;
4) A step of deriving the liposome from the microchannel of the deriving portion.
4). 4. The method for producing a liposome according to any one of items 1 to 3, wherein the microchannel has an inner diameter of 100 to 1000 μm.
5. Phospholipid dispersion liquid flow rate: The manufacturing method of the liposome of any one of the preceding clauses 1-4 whose gas flow rate is 0.001-10: 1.
6). 6. The method for producing a liposome according to any one of 1 to 5 above, wherein the gas flow rate is 1 to 15 ml / hour.
7). 7. The method for producing a liposome according to any one of items 1 to 6, wherein the temperature at the time of liposome formation is 15 to 80 ° C.
8). 8. The method for producing a liposome according to any one of 1 to 7 above, wherein an inclusion agent is further introduced into the microchannel, and the phospholipid dispersion, the encapsulation agent, and the gas are mixed in the microchannel. A method for producing a liposome containing an encapsulating agent.
9. 9. A liposome obtained by the method for producing a liposome according to any one of 1 to 8 above.
10. 10. The device for producing liposomes according to 9 above, comprising a microchannel.
11. 21. A liposome production kit comprising the liposome production device according to item 10 above, and a phospholipid dispersion not containing an organic solvent, an encapsulant and / or a gas introduction syringe.
本発明のリポソーム製剤の製造方法によれば、有機溶媒を使用することなく、さらに炭酸ガス等による加圧濾過や、有機溶媒を除去するための加熱処理、吸引処理や乾燥処理を行うことなく、粒子径が均一化されたリポソームを容易に製造しうる。 According to the method for producing a liposome preparation of the present invention, without using an organic solvent, without performing filtration under pressure with carbon dioxide gas or the like, heat treatment for removing the organic solvent, suction treatment or drying treatment, Liposomes having a uniform particle size can be easily produced.
本発明のリポソーム製剤の製造方法によれば、気体とリン脂質分散液との気液界面にリン脂質を吸着させて、配向を行うことができる。背景技術の欄で述べた特許文献１に記載の方法では、有機溶媒に溶解させたリン脂質溶液をマイクロ流路に導入し、有機溶媒を乾燥させてマイクロ流路の壁にリン脂質の薄膜を形成させて、リポソームを生成させており、壁面にリン脂質成分を吸着させるために有機溶媒が必要であった。一方、本発明の製造方法によれば、有機溶媒を必要とせず気液界面にリン脂質を吸着させることができ、リポソームを生成することができる。有機溶媒は、一般に毒性が問題となるので、完全に除去することが必須であったが、本発明の方法によれば、有機溶媒を用いないことにより、有機溶媒の残存可能性を完全に否定することができる。 According to the method for producing a liposome preparation of the present invention, orientation can be performed by adsorbing phospholipids to the gas-liquid interface between the gas and the phospholipid dispersion. In the method described in Patent Document 1 described in the background art section, a phospholipid solution dissolved in an organic solvent is introduced into a microchannel, and the organic solvent is dried to form a phospholipid thin film on the wall of the microchannel. It was formed to produce liposomes, and an organic solvent was required to adsorb the phospholipid component on the wall surface. On the other hand, according to the production method of the present invention, phospholipids can be adsorbed on the gas-liquid interface without using an organic solvent, and liposomes can be produced. The organic solvent generally has a problem of toxicity, so it was essential to remove it completely. However, according to the method of the present invention, the possibility of the organic solvent remaining is completely denied by not using the organic solvent. can do.
以下、本発明のリポソームの製造方法について詳細に説明する。本発明のリポソームの製造方法は、リポソームの生成工程において、マイクロ流路に、有機溶媒を含まない溶液とリン脂質成分からなるリン脂質分散液、及び気体を導入し、当該マイクロ流路内でリン脂質分散液と気体を混合させる工程を含むことを特徴とするリポソームの製造方法に関する。 Hereinafter, the production method of the liposome of the present invention will be described in detail. In the liposome production process of the present invention, in the liposome production step, a solution containing no organic solvent and a phospholipid dispersion liquid composed of a phospholipid component and a gas are introduced into the microchannel, and the phosphorous in the microchannel. The present invention relates to a method for producing a liposome, comprising a step of mixing a lipid dispersion and a gas.
本発明において「有機溶媒を含まない溶液」とは、有機溶剤を含まない水性媒体であって、リン脂質成分を分散しうる媒体をいい、特に限定されないが、例えば水、好適には注射用蒸留水、生理的食塩水、イオン交換水や、これらの溶液にグリセリン、グルコース、サッカロース、マルトースなどの等張化剤としての多価アルコールを０．２〜０．３Ｍ程度含有する水性媒体が挙げられる。本発明の製造方法によれば、製造時において有機溶媒、グリチルリチン類やコール酸類等のリポソームの形成助剤を用いることなく、簡便かつ効率良くリポソームを形成し得る利点を有する。また、本発明の有機溶媒を含まない溶液は、リン脂質成分の他、リポソームに内包されうる所望の内包剤を溶解しうる水性媒体、例えばリン酸緩衝液（ＰＢＳ）等であってもよい。また、本発明において「気体」とは、空気、不活性ガス、又はそれらの混合物を主成分とする気体が挙げられる。 In the present invention, the “solution not containing an organic solvent” refers to an aqueous medium that does not contain an organic solvent and can disperse a phospholipid component, and is not particularly limited. For example, water, preferably distilled for injection. Water, physiological saline, ion-exchanged water, and aqueous solutions containing about 0.2 to 0.3 M of polyhydric alcohol as an isotonic agent such as glycerin, glucose, saccharose, maltose, etc. are included in these solutions. . The production method of the present invention has an advantage that liposomes can be easily and efficiently formed without using liposome forming aids such as organic solvents, glycyrrhizins and cholic acids during production. In addition to the phospholipid component, the solution containing no organic solvent of the present invention may be an aqueous medium that can dissolve a desired encapsulating agent that can be encapsulated in liposomes, such as a phosphate buffer (PBS). In the present invention, the “gas” includes a gas mainly composed of air, an inert gas, or a mixture thereof.
本発明の製造方法で使用される「リン脂質成分」は、中性リン脂質として、大豆、卵黄などから得られるレシチン、リゾレシチン及び／又はこれらの水素添加物、水酸化物の誘導体を挙げることができる。これらは単独でも併用してもよい。その他のリン脂質として、卵黄、大豆又はその他、動植物に由来するホスファチジルコリン、ホスファチジルセリン、ホスファチジルイノシトール、ホスファチジルグリセロール、ホスファチジルエタノールアミン、スフィンゴミエリン、合成により得られるホスファチジン酸、ジパルミトイルホスファチジルコリン（DPPC）、ジステアロイルホスファチジルコリン（DSPC）、ジミリストリルホスファチジルコリン（DMPC）、ジオレイルホスファチジルコリン（DOPC）、ジパルミトイルホスファチジルグリセロール（DPPG）、ジステアロイルホスファチジルセリン（DSPS）、ジステアロイルホスファチジルグリセロール（DSPG）、ジパルミトイルホスファチジルイノシトール（DPPI）、ジステアロイルホスファチジルイノシトール（DSPI）、ジパルミトイルホスファチジン酸（DPPA）、ジステアロイルホスファチジン酸（DSPA）などを挙げることができる。 Examples of the “phospholipid component” used in the production method of the present invention include neutral phospholipids such as lecithin, lysolecithin and / or hydrogenated products and hydroxide derivatives thereof obtained from soybeans, egg yolks, and the like. it can. These may be used alone or in combination. Other phospholipids include egg yolk, soybean or other phosphatidylcholine derived from animals and plants, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, sphingomyelin, synthetically obtained phosphatidic acid, dipalmitoylphosphatidylcholine (DPPC), distearoyl Phosphatidylcholine (DSPC), Dimyristolphosphatidylcholine (DMPC), Dioleylphosphatidylcholine (DOPC), Dipalmitoylphosphatidylglycerol (DPPG), Distearoylphosphatidylserine (DSPS), Distearoylphosphatidylglycerol (DSPG), Dipalmitoylphosphatidylinositol (DPPI) ), Distearoyl phosphatidylinositol (DSPI), dipalmitoy Phosphatidic acid (DPPA), distearoyl lysophosphatidic acid (DSPA), and the like.
これらのリン脂質成分は通常、単独で使用されるが、２種以上併用して混合使用してもよい。２種以上の荷電リン脂質成分を使用する場合には、負電荷のリン脂質同士又は正電荷のリン脂質同士で使用することが、リポソームの凝集防止の観点から望ましい。 These phospholipid components are usually used alone, but two or more of them may be used in combination. When two or more kinds of charged phospholipid components are used, it is desirable to use negatively charged phospholipids or positively charged phospholipids from the viewpoint of preventing aggregation of liposomes.
本発明の「リン脂質分散液」には、所望により上記リン脂質成分の他に、他の成分を加えることもできる。その例として、膜安定化剤又はポリアルキレンオキシド基導入用アンカーとしてコレステロール、コレステロールエステルなどのステロール類、荷電物質であるジセチルホスフェートといったリン酸ジアルキルエステルなどが例示される。 In addition to the above phospholipid component, other components can be added to the “phospholipid dispersion” of the present invention as desired. Examples thereof include sterols such as cholesterol and cholesterol ester as membrane stabilizers or anchors for introducing polyalkylene oxide groups, and dialkyl phosphates such as dicetyl phosphate which is a charged substance.
本発明において「マイクロ流路」とは、導入部と導出部を有する、長さが２ｍｍ〜５ｍの流路であり、その流路内径は１００〜１０００μｍ、好ましくは２００〜５３０μｍである。流路内径は、所望するリポソーム粒子径により適宜調整されたものを使用することができる。本発明のマイクロ流路には、導入部に少なくとも２つのマイクロ流路を有し、導出部に１つのマイクロ流路を有する分岐型マイクロ流路であるのが好適である（図１参照）。例えば、導入部における１のマイクロ流路よりリン脂質分散液を導入し、導入部における他の１のマイクロ流路より気体を導入し、統合されたマイクロ流路内でリン脂質分散液と気体を混合させることができる。 In the present invention, the “micro flow path” is a flow path having a length of 2 mm to 5 m having an introduction part and a lead-out part, and the flow path inner diameter is 100 to 1000 μm, preferably 200 to 530 μm. The channel inner diameter can be appropriately adjusted according to the desired liposome particle diameter. The microchannel of the present invention is preferably a branched microchannel having at least two microchannels in the introduction part and one microchannel in the lead-out part (see FIG. 1). For example, a phospholipid dispersion is introduced from one microchannel in the introduction part, a gas is introduced from the other microchannel in the introduction part, and the phospholipid dispersion and gas are introduced into the integrated microchannel. Can be mixed.
マイクロ流路が、導入部に少なくとも２つのマイクロ流路を有し、導出部に１つのマイクロ流路を有する分岐型マイクロ流路の場合、本発明のリポソームは、以下の工程を含む製造方法により、製造することができる（図１参照）。
１）導入部における１のマイクロ流路よりリン脂質分散液を導入する工程；
２）導入部における他の１のマイクロ流路より気体を導入する工程；
３）マイクロ流路内で、リン脂質分散液と気体を混合させる工程；
４）導出部のマイクロ流路よりリポソームを導出する工程。When the microchannel is a branched microchannel having at least two microchannels in the introduction part and one microchannel in the lead-out part, the liposome of the present invention is produced by a production method including the following steps. Can be manufactured (see FIG. 1).
1) a step of introducing a phospholipid dispersion from one microchannel in the introduction part;
2) a step of introducing a gas from the other one microchannel in the introduction section;
3) mixing the phospholipid dispersion and gas in the microchannel;
4) A step of deriving the liposome from the microchannel of the deriving portion.
上記において、工程１）及び工程２）は同時に行うことができ、各々別の導入部よりリン脂質分散液及び気体をマイクロ流路内に導入することができる。導入部に３以上のマイクロ流路を有する場合は、その他のマイクロ流路より、水性媒体（有機溶媒を含まない溶液）を導入したり、内包剤を含む水性媒体を導入することができる。内包剤は、リン脂質分散液に混合して使用してもよい。 In the above, step 1) and step 2) can be performed simultaneously, and the phospholipid dispersion and gas can be introduced into the microchannel from different introduction portions. When the introduction part has three or more microchannels, an aqueous medium (solution not containing an organic solvent) or an aqueous medium containing an encapsulating agent can be introduced from other microchannels. The encapsulating agent may be mixed with the phospholipid dispersion and used.
上記において、工程１）及び工程２）において、リン脂質分散液及び気体をマイクロ流路内に導入する方法は、特に限定されないが、例えばリン脂質分散液又は気体を含むシリンジを、各々導入部のマイクロ流路に結合させて、ピストン又はポンプ等の作用によって導入することができる（図１参照）。リン脂質分散液及び気体をマイクロ流路内に導入させうる方法であれば、自体公知の方法であってもよいし、今後開発されるあらゆる方法を適用することができる。 In the above, the method for introducing the phospholipid dispersion and gas into the microchannel in step 1) and step 2) is not particularly limited. For example, a syringe containing the phospholipid dispersion or gas is used for each of the introduction parts. It can couple | bond with a microchannel and can introduce | transduce by the effect | action of a piston or a pump (refer FIG. 1). Any method known per se may be used as long as the phospholipid dispersion and gas can be introduced into the microchannel, and any method developed in the future can be applied.
上記において使用可能な内包剤は水溶性であり、脂質二重膜に囲まれた閉鎖空間の水相に内包される。内包剤の具体例として、広く医薬品に使用される水溶性の物質が挙げられる。具体的には造影剤、抗がん剤、抗酸化剤、抗菌剤、抗炎症剤、血行促進剤、美白剤、肌荒れ防止剤、老化防止剤、発毛促進剤、保湿剤、ホルモン剤、ビタミン類、色素、及びタンパク質類などが挙げられる。この中でもヒドロキシル基、カルボキシ基を分子構造中に含有する化合物が安定性の観点から好ましい。内包剤は、リン脂質分散液及び気体と同時にマイクロ流路内に導入してもよいし、リン脂質分散液及び気体をマイクロ流路内に導入した後に導入してもよい。 The encapsulating agent that can be used in the above is water-soluble, and is encapsulated in an aqueous phase in a closed space surrounded by a lipid bilayer membrane. Specific examples of the encapsulating agent include water-soluble substances widely used in pharmaceuticals. Specifically, contrast agents, anticancer agents, antioxidant agents, antibacterial agents, anti-inflammatory agents, blood circulation promoters, whitening agents, rough skin prevention agents, anti-aging agents, hair growth promoters, moisturizers, hormone agents, vitamins Class, dyes, and proteins. Among these, a compound containing a hydroxyl group or a carboxy group in the molecular structure is preferable from the viewpoint of stability. The encapsulating agent may be introduced into the microchannel simultaneously with the phospholipid dispersion and gas, or may be introduced after the phospholipid dispersion and gas are introduced into the microchannel.
上記において、リン脂質分散液におけるリン脂質と水性媒体の混合比は、リン脂質が溶解される十分量であれば特に限定されるものではないが、一般的には２〜３０ｍｇ／ｍｌの濃度に調整され、使用される。 In the above, the mixing ratio of the phospholipid and the aqueous medium in the phospholipid dispersion is not particularly limited as long as it is a sufficient amount to dissolve the phospholipid, but generally the concentration is 2 to 30 mg / ml. Regulated and used.
マイクロ流路に導入されるリン脂質分散液は、マイクロ流路の体積に応じて流路が十分に満たされる量であればよい。また、導入されるリン脂質分散液流量と気体流量の比は、リン脂質分散液流量：気体流量が、０．００１〜１０：１であり、好ましくは０．００１〜４：１であり、最も好ましくは、０．５〜２：１である。導入される気体流量は、１〜１５ｍｌ／時であり、好ましくは５〜１５ｍｌ／時であり、最も好ましくは５〜１０ｍｌ／時である。リン脂質分散液の導入速度は、流速が、０．０００１〜０．５ｍ／秒、好ましくは０．０５〜０．３ｍ／秒、より好ましくは０．１〜０．２ｍ／秒である。流路内の水溶液温度は、１５〜８０℃、好ましくは４５〜６５℃、より好ましくは５５〜６２℃である。 The amount of the phospholipid dispersion introduced into the microchannel may be an amount that can sufficiently fill the channel according to the volume of the microchannel. The ratio of the phospholipid dispersion flow rate and the gas flow rate introduced is such that the phospholipid dispersion flow rate: gas flow rate is 0.001 to 10: 1, preferably 0.001 to 4: 1. Preferably, it is 0.5-2: 1. The gas flow rate introduced is 1-15 ml / hour, preferably 5-15 ml / hour, most preferably 5-10 ml / hour. The introduction speed of the phospholipid dispersion is such that the flow rate is 0.0001 to 0.5 m / second, preferably 0.05 to 0.3 m / second, and more preferably 0.1 to 0.2 m / second. The aqueous solution temperature in the flow path is 15 to 80 ° C, preferably 45 to 65 ° C, more preferably 55 to 62 ° C.
上記条件で、気体とリン脂質分散液との気液界面にリン脂質を吸着させて、リン脂質の配向を行うことができる。背景技術の欄で述べた特許文献１には、有機溶媒に溶解させたリン脂質溶液をマイクロ流路に導入し、有機溶媒を乾燥させてマイクロ流路の壁にリン脂質の薄膜を形成させて、リポソームを生成させており、壁面にリン脂質成分を吸着させるために有機溶媒が必要であったのに対し、本発明の製造方法によれば、有機溶媒を必要とせず気液界面にリン脂質を吸着させることができ、リポソームを生成することができる（図２〜４参照）。 Under the above conditions, the phospholipid can be oriented by adsorbing the phospholipid to the gas-liquid interface between the gas and the phospholipid dispersion. In Patent Document 1 described in the Background Art section, a phospholipid solution dissolved in an organic solvent is introduced into a microchannel, and the organic solvent is dried to form a phospholipid thin film on the wall of the microchannel. In contrast to the production of liposomes, an organic solvent was required to adsorb the phospholipid component on the wall, whereas according to the production method of the present invention, the organic solvent was not required and the phospholipid was formed at the gas-liquid interface. Can be adsorbed and liposomes can be produced (see FIGS. 2 to 4).
本発明のリポソームの製造方法によれば、生成工程において、炭酸ガスによる加圧濾過工程を含まないでリポソームを製造することができる。 According to the method for producing liposomes of the present invention, liposomes can be produced without including a pressure filtration step using carbon dioxide gas in the production step.
本発明は、リポソームの製造用デバイスにも及ぶ。本発明のリポソームの製造用デバイスには、マイクロ流路が含まれる（図１参照）。当該マイクロ流路は、上記説明したように、導入部と導出部を有する長さが２ｍｍ〜５ｍの流路であり、その流路内径は１００〜１０００μｍ、好ましくは２００〜５３０μｍである。流路内径は、所望するリポソーム粒子径により、適宜調整される。本発明のリポソームの製造用デバイスに含まれるマイクロ流路には、導入部に少なくとも２つのマイクロ流路を有し、導出部に１つのマイクロ流路を有する分岐型マイクロ流路であるのが好適である。 The invention also extends to devices for the production of liposomes. The device for producing liposomes of the present invention includes a microchannel (see FIG. 1). As described above, the microchannel is a channel having a length of 2 mm to 5 m having an introduction part and a lead-out part, and the inner diameter of the channel is 100 to 1000 μm, preferably 200 to 530 μm. The inner diameter of the channel is appropriately adjusted depending on the desired liposome particle diameter. The microchannel included in the liposome production device of the present invention is preferably a branched microchannel having at least two microchannels in the introduction part and one microchannel in the lead-out part. It is.
本発明は、さらに、リポソームの製造用デバイスと導入用シリンジを含むリポソーム製造用キットにも及ぶ。当該導入用シリンジには、有機溶媒を含まないリン脂質分散液、内包剤及び／又は気体を充填し、これらの物質をマイクロ流路に導入させることができる。有機溶媒を含まないリン脂質分散液や内包剤は、用時調製することができる。また、有機溶媒を含まないリン脂質分散液に、内包剤を加え、同時にマイクロ流路内に導入することもできる。 The present invention further extends to a liposome production kit including a liposome production device and an introduction syringe. The introduction syringe can be filled with a phospholipid dispersion containing no organic solvent, an encapsulant and / or a gas, and these substances can be introduced into the microchannel. A phospholipid dispersion and an encapsulating agent not containing an organic solvent can be prepared at the time of use. In addition, an inclusion agent can be added to a phospholipid dispersion containing no organic solvent and simultaneously introduced into the microchannel.
本発明は、上記製造方法により得られたリポソームにも及ぶ。本発明の製造方法により生成されたリポソームは、マイクロ流路の導出部から排出される。生成されたリポソームは、遠心分離等の通常の手段で沈殿として回収される。回収されたリポソームは、マイクロ流路の内径により、粒径が均一化されているが、所望により粒子径を調整するためのろ過を行ってもよい。また、製剤化のための付加処理、滅菌等が行われ、所望により凍結乾燥化及び／又は水溶液製剤として調製される。 The present invention also extends to liposomes obtained by the above production method. Liposomes produced by the production method of the present invention are discharged from the outlet of the microchannel. The produced liposomes are collected as a precipitate by ordinary means such as centrifugation. The collected liposomes have a uniform particle size depending on the inner diameter of the microchannel, but may be filtered to adjust the particle diameter as desired. Further, addition treatment for preparation, sterilization and the like are performed, and if desired, lyophilized and / or prepared as an aqueous solution preparation.
本発明の製造方法によって製造されるリポソームとしては、多重層膜からなるリポソーム（Multilamellar vesicles; ＭＬＶ）、粒子径が大きい一枚膜のＬＵＶからなるリポソーム（Large unilamellar veislcles）、粒子径が５０ｎｍ未満の小さい一枚膜のＳＵＶからなるリポソーム（Small unilamellar vesicles）が挙げられ、これらは混在していてもよいが、本発明の方法によれば粒径がほぼ均一化したリポソームを得ることができる。 As the liposome produced by the production method of the present invention, a liposome composed of multilamellar vesicles (MLV), a liposome composed of a single membrane LUV having a large particle diameter (Large unilamellar veislcles), a particle diameter of less than 50 nm Liposomes (Small unilamellar vesicles) composed of small single-film SUVs may be mentioned, and these may be mixed, but according to the method of the present invention, liposomes having a substantially uniform particle size can be obtained.
また、本発明の製造方法によって製造されるリポソームは、リポソームの水性分散液剤又は凍結乾燥剤として調製することができる。内包剤を含有するリポソームとして好適な製剤は、内包剤が水溶性の薬剤であり、水溶性薬剤が内包されたリポソームが水性溶媒中に分散している水性分散液製剤が挙げられる。このリポソームの水性分散液剤には、安定化剤、キレート剤、抗酸化剤、粘度調節剤、緩衝剤、ｐＨ調整剤などが溶解又は分散していてもよい。このようなリポソームの水性分散液製剤の調製は、各種の方法が知られているが、目的、特性、用途などに応じて適宜選択して行う。 In addition, the liposome produced by the production method of the present invention can be prepared as an aqueous dispersion or lyophilization agent of the liposome. The preparation suitable as the liposome containing the encapsulating agent includes an aqueous dispersion formulation in which the encapsulating agent is a water-soluble drug and the liposome encapsulating the water-soluble drug is dispersed in an aqueous solvent. In the liposome aqueous dispersion, stabilizers, chelating agents, antioxidants, viscosity modifiers, buffers, pH adjusters and the like may be dissolved or dispersed. Various methods are known for preparing such an aqueous dispersion formulation of liposome, and it is appropriately selected according to the purpose, characteristics, use and the like.
以下、実施例によって本発明をさらに具体的に説明するが、本発明はこれらの実施例に限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, this invention is not limited to these Examples.
（実施例１）リポソームの製造
本発明のリポソームを、図１に示すマイクロ流路を含むリポソーム製造用デバイスを用いて製造した。
卵黄レシチン０．００２ｇをイオン交換水１ｍｌに完全に分散するまで攪拌し、リン脂質分散液を調製した。導入するマイクロ流路（デバイス）及びリン脂質分散液を６０℃に保ち、内径２７０μｍ、長さ１０ｃｍの３分岐からなる流路の導入部にリン脂質分散液、空気及びＰＢＳを導入した。空気は流量１〜１５ｍｌ／時で導入し、同時にリン脂質分散液及びＰＢＳを流速０．０００１〜０．５ｍ／秒で導入した。マイクロ流路の導出部から形成されたリポソームの分散水溶液を得た。この水溶液を１２０００ｒｐｍの条件で遠心分離し、多重膜リポソームを沈殿として回収した。沈殿を凍結乾燥し、乾燥リポソーム製剤を製造した。(Example 1) Manufacture of liposomes The liposomes of the present invention were manufactured using a liposome manufacturing device including the microchannel shown in Fig. 1.
A phospholipid dispersion was prepared by stirring until 0.002 g of egg yolk lecithin was completely dispersed in 1 ml of ion exchange water. The microchannel (device) to be introduced and the phospholipid dispersion were kept at 60 ° C., and the phospholipid dispersion, air and PBS were introduced into the introduction portion of the three-branch channel having an inner diameter of 270 μm and a length of 10 cm. Air was introduced at a flow rate of 1 to 15 ml / hour, and at the same time, a phospholipid dispersion and PBS were introduced at a flow rate of 0.0001 to 0.5 m / second. A dispersed aqueous solution of liposomes formed from the outlet of the microchannel was obtained. This aqueous solution was centrifuged at 12000 rpm, and multilamellar liposomes were collected as a precipitate. The precipitate was lyophilized to produce a dry liposome preparation.
本発明の製造方法によれば、分岐型マイクロ流路の合流部において図２に示す流れが認められた。また、流路内の様子を図３に、気液界面にリン脂質を吸着させている状態を図４Ａ、Ｂに示した。 According to the manufacturing method of the present invention, the flow shown in FIG. 2 was observed at the junction of the branched microchannel. FIG. 3 shows the state in the flow path, and FIGS. 4A and 4B show the state in which the phospholipid is adsorbed on the gas-liquid interface.
（実験例１）粒度分布１
実施例１で製造したリポソーム及び比較例としてのバンガム法（非特許文献１）で製造したリポソームの粒子径を測定し、その結果を図５に示した。(Experimental example 1) Particle size distribution 1
The particle diameters of the liposomes produced in Example 1 and the liposomes produced by the Bangham method (Non-Patent Document 1) as a comparative example were measured, and the results are shown in FIG.
リポソーム分散水溶液を流路出口で回収し、これを１２００ｒｐｍで１０分間遠心分離し、多重膜リポソームの沈殿として回収した。その後沈殿を生理食塩水１ミリリットル中に分散させ、得られた溶液に含まれるリポソームの粒子径を、動的光散乱法を用いて測定した。 The liposome-dispersed aqueous solution was recovered at the outlet of the flow path, and centrifuged at 1200 rpm for 10 minutes to recover as a multilamellar liposome precipitate. Thereafter, the precipitate was dispersed in 1 ml of physiological saline, and the particle size of the liposome contained in the obtained solution was measured using a dynamic light scattering method.
図５において横軸はリポソームの粒子径（ｎｍ）、縦軸は確率密度(intensity)（％）を示した。実線は本願発明、破線はバンガム法によるものである。比較例としてのバンガム法によるとブロードな粒子径分布により各種粒子径のリポソームが生成されたのに対し、本発明の製造方法によると、１つの鋭いピークからなる粒子径分布が認められ、均一化された粒子径のリポソームが生成されることが確認された。 In FIG. 5, the horizontal axis represents the liposome particle size (nm), and the vertical axis represents the probability density (%). The solid line is based on the present invention, and the broken line is based on the Bangham method. According to the bangham method as a comparative example, liposomes with various particle sizes were generated by a broad particle size distribution, whereas according to the production method of the present invention, a particle size distribution consisting of one sharp peak was recognized and homogenized. It was confirmed that liposomes having the specified particle size were produced.
（実験例２）粒度分布２
実施例１のリポソームの製造方法において、空気を含む条件及び空気を含まない条件で、リポソームを製造した。空気を含む場合のリン脂質溶液及びＰＢＳの流量は１６．７μｌ／分、空気流量は５ｍｌ／時であり、空気を含まない場合のリン脂質溶液及びＰＢＳの流量は１５μｌ／分であった。(Experimental example 2) Particle size distribution 2
In the liposome production method of Example 1, liposomes were produced under conditions including air and not including air. The flow rate of the phospholipid solution and PBS when air was included was 16.7 μl / min, the air flow rate was 5 ml / hour, and the flow rate of phospholipid solution and PBS when air was not included was 15 μl / min.
得られたリポソームについて、実験例１と同手法により粒子径を測定した。その結果、空気を含む条件下で製造したリポソームでは１つの鋭いピークからなる粒子径分布が認められ、均一化された粒子径のリポソームが生成されることが確認された（図６）。 About the obtained liposome, the particle diameter was measured by the same method as Experimental Example 1. As a result, in the liposome produced under conditions containing air, a particle size distribution consisting of one sharp peak was observed, and it was confirmed that liposomes with a uniform particle size were produced (FIG. 6).
（実験例３）収率
実施例１に示すリポソームの製造方法において、リン脂質分散液とＰＢＳの流量及び空気流量を変えたときのリポソームの収率を測定した。収率は、用いたリン脂質（重量）に対するリポソームを構成するリン脂質（重量）の重量比（％）により算出した。(Experimental Example 3) Yield In the liposome production method shown in Example 1, the yield of liposome was measured when the flow rate of phospholipid dispersion and PBS and the air flow rate were changed. The yield was calculated by the weight ratio (%) of the phospholipid (weight) constituting the liposome to the phospholipid (weight) used.
リン脂質分散液とＰＢＳの流量及び空気流量を変えたときのリポソームの収率を図７に示した。なお、比較例としてバンガム法で製造したリポソームの収率を破線で示した。その結果、本発明の製造方法によれば、バンガム法で製造した場合に比べて優れた収率を示した。また、空気流量に対するリン脂質分散液とＰＢＳの流量比と収率を図８に示した。その結果、空気流量が、１〜１５ｍｌ／時であって、リン脂質分散液流量：空気流量が、０．００１〜１０：１の場合に、高い収率でリポソームを得ることができた。より具体的には、空気流量が１０ｍｌ／時より多い場合は、空気流量に対するリン脂質分散液とＰＢＳの流量比が１付近で最も収率が高く、空気流量が５ｍｌ／時より少ない場合は、空気流量に対するリン脂質分散液とＰＢＳの流量比が大きいほうが収率が高かった（図８）。 FIG. 7 shows the yield of liposomes when the flow rate of phospholipid dispersion and PBS and the air flow rate were changed. In addition, the yield of the liposome manufactured by the bangham method as a comparative example was shown with the broken line. As a result, according to the production method of the present invention, an excellent yield was obtained as compared with the case of production by the Bangham method. Moreover, the flow rate ratio and yield of the phospholipid dispersion and PBS with respect to the air flow rate are shown in FIG. As a result, when the air flow rate was 1 to 15 ml / hour and the phospholipid dispersion flow rate: air flow rate was 0.001 to 10: 1, liposomes could be obtained with a high yield. More specifically, when the air flow rate is higher than 10 ml / hour, the yield is highest when the flow rate ratio between the phospholipid dispersion and the PBS with respect to the air flow rate is near 1, and when the air flow rate is less than 5 ml / hour, The yield was higher when the flow rate ratio of the phospholipid dispersion and PBS to the air flow rate was larger (FIG. 8).
以上詳述したように、本発明の有機溶媒を使用しない方法でリポソームを製造することにより、加圧濾過処理や、有機溶媒を除去するための加熱処理、吸引処理や乾燥処理などの工程を省略することができ、リポソーム製造に要する時間の短縮化ができ、さらに従来よりも高収率でリポソームを製造することできる。リポソームの寿命は、通常３日程度であるといわれているが、本発明の製造方法によれば、コンパクトなリポソーム製造用キット又は製造用装置により、短時間に所望の内包剤を含むリポソーム製剤を製造することが可能となり、病院や薬局等でのリポソーム製剤を製造することができ、安定な状態で使用することができる。 As described in detail above, by producing liposomes by a method that does not use the organic solvent of the present invention, steps such as pressure filtration treatment, heat treatment for removing the organic solvent, suction treatment and drying treatment are omitted. The time required for liposome production can be shortened, and liposomes can be produced in higher yields than in the past. The lifespan of liposomes is usually said to be about 3 days. However, according to the production method of the present invention, a liposome preparation containing a desired encapsulating agent can be obtained in a short time by using a compact liposome production kit or production device. It becomes possible to manufacture, and it is possible to manufacture liposome preparations in hospitals, pharmacies and the like, and it can be used in a stable state.
このリポソームは、内部に有する水相には水溶性の内包剤（例えば、薬効成分）を、二分子膜の内部には油溶性の内包剤を保持するという、いわゆるカプセル構造を構築することができる。本発明の製造方法により製造したリポソームにより、検査（診断）、治療、化粧などの様々な分野で用いることができる。さらに、近年では、薬物送達システム（ＤＤＳ）への応用が盛んに研究されており、本発明の製造方法により、リポソーム製剤の普及に対して多いに貢献することができる。 This liposome can construct a so-called capsule structure in which a water-soluble inclusion agent (for example, a medicinal component) is retained in the aqueous phase inside and an oil-soluble inclusion agent is retained in the bilayer membrane. . The liposome produced by the production method of the present invention can be used in various fields such as examination (diagnosis), treatment, and makeup. Furthermore, in recent years, application to a drug delivery system (DDS) has been actively studied, and the production method of the present invention can greatly contribute to the spread of liposome preparations.
Claims (8)
１）導入部における１のマイクロ流路よりリン脂質分散液を導入する工程；
２）導入部における他の１のマイクロ流路より気体を導入する工程；
３）マイクロ流路内で、リン脂質分散液と気体を混合させる工程；
４）導出部のマイクロ流路よりリポソームを導出する工程。 The micro-channel is a branched micro-channel having at least two micro-channels in the introduction portion and one micro-channel in the lead-out portion, and includes the following steps. The method for producing the liposome according to 1 or 2:
1) a step of introducing a phospholipid dispersion from one microchannel in the introduction part;
2) a step of introducing a gas from the other one microchannel in the introduction section;
3) mixing the phospholipid dispersion and gas in the microchannel;
4) A step of deriving the liposome from the microchannel of the deriving portion.
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