WO2003000291A1 - Lipid vesicles, process for producing lipid vesicles and method of immobilizing gene on lipid vesicles - Google Patents

Lipid vesicles, process for producing lipid vesicles and method of immobilizing gene on lipid vesicles Download PDF

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Publication number
WO2003000291A1
WO2003000291A1 PCT/JP2002/006312 JP0206312W WO03000291A1 WO 2003000291 A1 WO2003000291 A1 WO 2003000291A1 JP 0206312 W JP0206312 W JP 0206312W WO 03000291 A1 WO03000291 A1 WO 03000291A1
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Prior art keywords
lipid
lipid vesicle
producing
gene
vesicle
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PCT/JP2002/006312
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French (fr)
Japanese (ja)
Inventor
Keiichi Kato
Norio Koine
Takuya Sugawara
Shigemitsu Takashima
Yuji Heike
Yoshio Hisaeda
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Techno Network Shikoku Co., Ltd.
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Priority claimed from JP2001190515A external-priority patent/JP2003012503A/en
Priority claimed from JP2001190518A external-priority patent/JP2003001097A/en
Priority claimed from JP2001266659A external-priority patent/JP4928689B2/en
Application filed by Techno Network Shikoku Co., Ltd. filed Critical Techno Network Shikoku Co., Ltd.
Publication of WO2003000291A1 publication Critical patent/WO2003000291A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • 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
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • the present invention relates to a lipid vesicle, a method for producing the lipid vesicle, and a method for immobilizing a gene on the lipid vesicle.
  • the present invention relates to a lipid vesicle (closed endoplasmic reticulum composed of a lipid molecular membrane) useful as a drug carrier and a method for immobilizing a gene to the lipid vesicle in DDS for cancer therapy or gene therapy.
  • a lipid vesicle closed endoplasmic reticulum composed of a lipid molecular membrane
  • ribosomes composed of natural phospholipids account for 50% or more (actually almost all) of the lipid membrane components, and these are the reverse-phase evaporation method or ultrasonic irradiation method. It has been prepared.
  • these ribosomes have the following problems: (1) the inclusion ratio of substances inside the ribosome is small; (2) the small volume of the internal aqueous phase of the ribosome makes it difficult to include macromolecules such as genes; (3) the stability of the ribosome is poor; There was a problem.
  • a method for producing a vesicle in which an artificial lipid membrane is composed of sonolebitan ester has been proposed, and is expected to be a stable and inexpensive carrier.
  • phagocytes such as macrophages in the body
  • ribosomes or vesicles are administered as carriers, they are phagocytosed (captured / degraded) by the phagocytes before reaching the affected area and acting.
  • lipid vesicles have a problem in that the outside has a negative polarity, and it is difficult to attach DNA, which is also a negative polarity. Therefore, conventional lipid vesicles have poor adhesion efficiency of DNA, and it is difficult to use them as gene carriers for gene therapy and the like.
  • An object of the present invention is to provide a lipid vesicle which is not easily captured by phagocytic cells and can be used as a carrier in DDS which enables effective chemical treatment, and a method for producing the same. Another objective is to give the lipid vesicle as a carrier the target directivity (missile function) for the target cell. Another object is to effectively fix genes on lipid vesicles. Disclosure of the invention
  • the method for producing a lipid vesicle according to the present invention includes: a step of adding water to a treatment liquid containing a sorbitan ester as a primary emulsifier and a solvent, and emulsifying the mixture by ultrasonic irradiation; and
  • This is a two-stage emulsification method that includes the step of distilling off and then emulsifying and emulsifying a secondary emulsifier with an ethylene oxide adduct of a sorbitan ester.
  • the method of producing a lipid vesicle may include a step of evaporating a solvent from the treated solution, adding a sorbitan ester ethylene oxide adduct as a secondary emulsifier to the remaining solution, and stirring and emulsifying the mixture.
  • —Sorbitan oleic acid monoester may be used as the secondary emulsifier, and sorbitan oleic acid monoester ethylene oxide adduct may be used as the secondary emulsifier.
  • Ultra sound while cooling the processing liquid during primary emulsification Wave irradiation may be performed.
  • the membrane may be subjected to membrane filtration to pass through a nano-sized lipid vesicle having a particle size of 200 nm or less.
  • a treatment liquid is prepared by adding water to a solvent in which a sorbitan ester as a primary emulsifier is dissolved, and the treatment liquid is cooled and subjected to ultrasonic irradiation while being emulsified.
  • a primary emulsification step in which the solvent is distilled off from the treated solution subjected to the above-mentioned treatment, and a secondary emulsifier in which an ethylene oxide adduct of a sorbitan ester and a PEG lipid are blended and emulsified as a secondary emulsifier. It is characterized by including a step.
  • the process solution may further include a step of mixing iotadecyl ester of isothiocyanic acid (IAOE) into the treatment solution, and a step of mixing soluble protein A after the secondary emulsification step and a step of adding an antibody.
  • IAOE isothiocyanic acid
  • the method for immobilizing a gene on a lipid vesicle comprises the steps of: adding a nucleic acid-binding protein to a plasmid gene; and mixing the gene with a lipid vesicle composed of an artificial lipid containing a cationic peptide lipid. This will be fixed.
  • the nucleic acid binding protein is preferably selected from the group consisting of histone, protamine and poly-L-lysine, and more preferably two or more of them are combined.
  • the lipid vesicle according to the present invention is one produced by the above-mentioned production method, or one obtained by immobilizing a gene by the above-mentioned method for immobilizing a gene. Further, the lipid vesicle according to the present invention comprises an artificial lipid containing a cationic peptide lipid, a plasmid gene, a plasmid gene, and a nucleic acid-binding agent selected from the group consisting of histamine, protamine and poly-L-lysine. It may have a protein.
  • Another lipid vesicle according to the present invention is sol ⁇
  • FIG. 1 is a cross-sectional view showing a step of primary emulsification in the production of a lipid vesicle of the present invention.
  • FIG. 2 is a graph showing the relationship between the ratio of lecithin and cholesterol in total lipids and the rate of vesicle formation.
  • FIG. 3 is a graph showing the relationship between the ratio of PEG lipids to the total lipids in the preparation of microsize vesicles and the production rate of vesicles.
  • FIG. 4 is a graph showing the relationship between the percentage of PEG lipids in the total lipids and the vesicle generation rate in the preparation of nano-sized vesicles.
  • FIG. 1 is a cross-sectional view showing a step of primary emulsification in the production of a lipid vesicle of the present invention.
  • FIG. 2 is a graph showing the relationship between the ratio of lecithin and cholesterol in total lipids and the rate of vesicle
  • FIG. 5 is an explanatory diagram of a method for immobilizing a gene according to the present invention.
  • FIG. 6 is a graph showing the result of applying the method for immobilizing a gene according to the present invention to a micropore-sized vesicle.
  • Figure 7 is a more detailed graph of the Daraf.
  • FIG. 8 is a graph showing the results of applying the method for immobilizing a gene according to the present invention to nano-sized vesicles.
  • FIG. 9 is a graph showing the result of applying the method of immobilizing a gene according to the present invention using a histone.
  • FIG. 10 is a graph showing the results of comparing various combinations of nucleic acid binding proteins.
  • Sorbitan oleate monoester (trade name) “Span 80 j manufactured by Wako Pure Chemical Industries, Ltd.) and lecithin and cholesterol previously dissolved in hexane were added dropwise while stirring with a homomixer, and ultrasonic irradiation was repeated using an ultrasonic homogenizer. Then, the WZO-type emulsion was placed in a flask, and hexane was removed under reduced pressure. The thus-prepared W / L-type emulsion contained ethylene oxide adduct of sorbitan oleic acid monoester ( An aqueous solution of “Tween 80” (product of Wako Pure Chemical Industries) may be stirred and mixed, and then centrifuged.
  • Teween 80 product of Wako Pure Chemical Industries
  • FIG. 1 is a cross-sectional view showing a primary emulsification process of this example.
  • Sorbitanoleic acid monoester (trade name “Span 80 J made by Wako Pure Chemical Industries, Ltd.”), which is the main component of the vesicle, in a test tube, inner container such as a test vessel, etc. 66 mg, and n-hexane 1 ml in advance Take Lecithin and Cholesterol, which were dissolved to a concentration of 5 mg, in the required amount (6 mg, 3 mg), and add treated solution 2 to which n-hexane was added so that the total amount was 3 ml.
  • the mixture is stirred for 30 seconds with a homomixer (15, 000 rpm), and while stirring the solution with a homomixer, an aqueous solution 3 (0.1. 5 ml) was added dropwise little by little (1 minute), and then ultrasonic irradiation was performed using an ultrasonic homogenizer 4. This ultrasonic irradiation was repeated three times, a cycle of 15 seconds and a rest of 15 seconds. ⁇ ⁇ Install outer container 5 outside container 1 and place ice between inner container 1 and outer container 5. Cooling the processing solution 2 by adding 6 suppresses the temperature rise of the processing solution 2 due to the ultrasonic irradiation and prevents the n-hexane in the processing solution 2 from igniting.
  • the output of (4) is appropriately selected depending on the amount of the processing solution 2 to be put into the inner container 1 and the target particle diameter of the vesicle, but it is necessary to obtain a nano size of 200 nm or less in a small vessel such as a test tube. Is suitable for a particle size of about 50 to 140 W, especially for a particle size of 100 nm or less. _
  • the wzo emulsion obtained in this manner is put into a NASA flask, depressurized by a rotary evaporator to remove n-hexane, and sorbitan oil is added to the W / L emulsion thus obtained.
  • 3 ml of an aqueous solution of an ethylene oxide adduct of an acid monoester [trade name “Twin 80” (l S mg Zm l) manufactured by Wako Pure Chemical Industries) was added, and the mixture was stirred using a homomixer (3000 rpm). Then, centrifugation was performed to prepare nano-sized lipid vesicles. The size of the nano vesicle was observed by a transmission electron microscope, and as a result, it was confirmed that the average particle size of the vesicle was 40 to 100 nm.
  • the lipid vesicles obtained in this manner are novel in themselves. That is, unlike conventional ribosomes and microphone-sized lipid vesicles, the size is as small as about 100 nm or less. It has a structure in which substances can be contained at a high inclusion rate. The size is relatively uniform and can be adjusted to some extent by changing the frequency, intensity, and duration of the ultrasonic irradiation. Furthermore, by performing membrane filtration, the size can be adjusted to the so-called nano size of 200 nm or less (especially, to less than 100 nm when sufficiently irradiated with ultrasonic waves) together with the sterilization treatment. The stability in physiological saline is good, and it remains stable even in serum except for about 30% of damage when injected. In addition, it was in good contact with the lymphocytes and the encapsulated substance was able to function.
  • the size of the nano vesicle was observed by a transmission electron microscope, and as a result, it was confirmed that the average particle size of the vesicle was 40 to 100 nm.
  • Sorbitanoleic acid monoester (trade name “Snod. 80”) Add 13 mg of lecithin to 12 mg of cholesterol and 12 mg of cholesterol mono-oleate, and add 0.1 mg of n-hexane to 4.0 mg of n-hexane. The mixture was dissolved in 0 ml and stirred with a microphone-mouth homogenizer for 30 seconds. Next, the mixture was stirred for 1 minute while adding 0.30 ml of an aqueous solution of the inclusion substance to be an internal aqueous phase, and then subjected to ultrasonic irradiation with an ultrasonic homogenizer to perform primary emulsification.
  • the treatment liquid was put in a NAS flask, and the pressure was reduced by a rotary evaporator to remove n- hexane.
  • lipid vesicle To prepare a nano-sized lipid vesicle.
  • the method for producing lipid vesicles according to the present invention can be applied on various scales, large and small, in particular, for relatively small and simple implementation, sorbitan esters 10 to 1 OOO mg, sonorebitan ester It is advisable to adjust the volume of water with ethylenoxide to the range of 10 to 50 mg, the internal water volume of the vesicles to 0.1 to 1 m1, and the external water volume to 1 to 10 m1.
  • the size of the nano vesicle was observed by a transmission electron microscope, and as a result, it was confirmed that the average particle diameter of the vesicle was about 40 to 100 nm.
  • the w / o emulsion was placed in a flask to remove hexane under reduced pressure, and the ethylene oxide of sorbitan monooleate monoester was added to the W / L emulsion thus prepared.
  • An aqueous solution of a PEG lipid having an adduct and a lipid derivative (trade name “SU NB RI GHT DSPE-20 HC J Nippon Oil & Fats Co., Ltd.”) may be mixed by stirring (secondary conversion step) and then centrifuged. .
  • the primary in emulsion Engineering degree may be due connexion stirring homomixer one instead of ultrasonic irradiation c below, a stealth by example
  • the method for producing a lipid vesicle will be described in more detail.
  • FIG. 1 shows the primary emulsification process of this example. Contents of test tubes, beakers, etc. ⁇
  • sorbitan oleate monoester (trade name “Span 80”, manufactured by Wako Pure Chemical Industries, Ltd.) and lecithin and cholesterol, which are the main components of the vesicle, into vessel 1 and dissolve in n-hexane (4 ml). This solution is stirred for 30 seconds with a homomixer (15, O O O rpm). Aqueous solution 3 (0.3 ml), which becomes the internal aqueous phase in which the inclusion substance in the vesicle was adjusted to a predetermined concentration, was added dropwise little by little while stirring with a homomixer for 1 minute, and then using an ultrasonic homogenizer 4. To irradiate ultrasonic waves.
  • This ultrasonic irradiation was repeated for 15 seconds and then rested for 15 seconds three times.
  • An outer container 5 is provided outside the inner container 1, and ice water 6 is put between the inner container 1 and the outer container 5 to cool the processing liquid 2, thereby suppressing the temperature rise of the processing liquid 2 due to ultrasonic irradiation. It prevents the n-hexane in the treatment liquid 2 from igniting.
  • the WZO emulsion thus obtained is placed in an eggplant-shaped flask, and the pressure is reduced by a rotary evaporator, and n-hexane is removed at 28.
  • the preparation of nano-sized vesicles has been described as an example.However, the above method can be applied to the preparation of micro-sized vesicles by stirring only with a homomixer without performing ultrasonic irradiation during primary emulsification. it can.
  • Sorbitan oleate monoester a commercial product, was used as sorbitan oleate monoester, which is the main component of the vesicle.
  • the sorbitan oleate monoester has a single fatty acid chain.
  • commercially available products contain impurities with two or more chains, it is better to use products containing these impurities than pure sorbitan. ⁇ 0
  • Fig. 3 shows the case of micro-sized vesicles
  • Fig. 4 shows the case of nano-sized vesicles.
  • the abscissa is the mole percentage of PEG lipid per span 80.
  • the vertical axis represents the vesicle formation rate.
  • the concentration of the fluorescent substance carboxyfluorescein (CF) contained in the aqueous solution dropped during the primary emulsification preparation of the vesicle and the final vesicle formation It is a numerical value expressed as the ratio of the concentration of CF taken into the water above the inner layer. From this, it can be seen that the generation rate is high and the optimum value is obtained when the PEG lipid content is about 2 mol% for span 80.
  • CF carboxyfluorescein
  • a method for immobilizing an antibody on a PEG lipid vesicle having a stealth function to impart target directivity will be described.
  • the production method of this example is also based on the production method of Example 1, but in order to further immobilize the antibody, I AOE which is a lipid anchor for immobilizing protein A and protein A for controlling the posture of the antibody And an operation of adding an antibody.
  • IAOE octadecyl isothiocyanate
  • lipid anchor I AOE (7.5 mg: 10% by weight based on the total fat mass) is mixed with span 80 (43.7 mg), which is the main component of the vesicle, and lecithin (0. 8 6 5 mg), cholester ⁇ 2
  • the antibody binds to protein A at a site (Fc portion) opposite to the antigen binding point, and this protein A is bound to the PEG lipid vesicle by the lipid anchor I AOE. It is firmly fixed on the surface.
  • Fc portion a site opposite to the antigen binding point
  • Fc portion a site opposite to the antigen binding point
  • Microphone-sized vesicles and nano-sized vesicles were prepared using the production method of this example, and were brought into contact with cancer cells.
  • the cells used were HB4C5 cells, which are hybridomas of lymphoma and lymphocytes derived from a lung cancer patient.
  • HB4C5 cells produce IgM antibodies on their cell surface that are antigens for anti-human IgM antibodies.
  • the lipid vesicles are prepared so that DNA-PI is contained in the water, and by observing the fluorescence generated by the action of DNA-PI after contact, the inclusion substance DNA-PI in the lipid vesicles is converted to HB It is possible to determine whether or not it has been taken into 4 C 5 cells.
  • ESA is a substance that acts on DNA of cancer cells and has an effect of inducing programmed death of cancer cells, that is, apoptosis.
  • a test was conducted in which cancer cells cultured in a vessel containing lipid vesicles with ESA immobilized were transplanted into the body of five experimental rats.
  • cancer cells cultured in a vessel containing lipid vesicles without immobilized ESA were transplanted to the body of an experimental rat, 5 .
  • pcDA3-1uci having a backbone of pcDA3 as a plasmid gene and a luciferase expression gene incorporated as a reporter gene was used.
  • the plasmid DNA was treated with a nucleic acid-binding protein at a plus reagent (combination of a nucleic acid-binding protein and a nuclear transport signal).
  • Vesicles were prepared as cationic lipid-containing lipid vesicles by mixing 20 to 30% by weight of cationic peptide lipid (CPL) with the constituent lipids.
  • lipid vesicle having a particle size of 1 ⁇ or more was used.
  • a mouth-sized vesicle was used.
  • Sorbitan oleic acid monoester is used as the main component.
  • the lipid vesicle according to the present invention is adjusted to have a CPL at a ratio of 30% by weight to the constituent lipid.
  • the resulting vesicle-containing solution is mixed with plasmid DNA by appropriately diluting with physiological saline, and the gene is immobilized on lipid vesicles, which are then transferred to the target cells, HeLa cells. Time acted. After 48 hours, a substrate substance was added, and the color development was measured. Since the gene contains a luciferase-expressing gene, the introduced gene functions and, when luciferase is expressed, reacts with a substrate substance to develop color.
  • Figures 6 and 7 show the results.
  • the vertical axis indicates the amount of luciferase activity, and a higher value indicates that the introduced gene is functioning and that the target cell has been transformed.
  • L'AMINE is a comparative example, and is a result of binding of liposome to plasmid treated with ribofectamine plus reagent.
  • span 80 vesicles is an experimental value of lipid vesicles not containing CPL. According to this, gene transfer was not sufficiently performed in vesicles without CPL.
  • vesicles containing CPL are well transduced and have a function equivalent to or better than that of liposomes by appropriate dilution.
  • the particle size as a lipid base consequent Le is shown in Figure 8 the t result with 1 OO nm or less called nano-sized base Shikuru . It can be seen that even a nano-sized vesicle functions as a gene carrier.
  • Target cells include MDAMB-468 (human breast cancer cells), CHO (Chinese hamster ovary cells), and COS 7 (cells derived from African monkey kidney) in addition to HeLa cells. In each case, a replacement paper was used as a gene carrier (Rule 26). , see
  • histone nucleic acid binding protein As a third embodiment of genes immobilization method according to the present invention, histone nucleic acid binding protein, protamine or poly-L-lysine c
  • nucleic acid binding protein that shows an example was used, as compared to Ribofueta Taminpurasu reagent Inexpensive and easy to obtain.
  • Fig. 9 shows the results when the histone is used. It can be seen that the effect is equal to or higher than that of the expensive ribofectamine plus reagent when the concentration is appropriately adjusted and used.
  • histone, protamine and poly-L-lysine are more effective when used in combination than when used alone.
  • Figure 10 shows the results of experiments with different combinations.
  • the lipid vesicle, the method for producing the lipid vesicle, and the method for immobilizing a gene on the lipid vesicle according to the present invention are inexpensive and stable for carrying a gene or drug effective for treating a disease such as cancer to an affected area. It can be formed with various artificial lipids and has high utility value in the pharmaceutical industry. Furthermore, it is hard to be trapped by phagocytic cells in the body and acts only on the affected area, such as cancer cells, avoiding normal cells. The scope of application in the pharmaceutical industry is wide.

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Abstract

Lipid vesicles having a small size can be obtained by a two-step emulsification process which comprises the step of adding water to a treatment liquor containing a sorbitan ester as a primary emulsifier and a solvent and emulsifying by ultrasonication and the step of distilling off the solvent from the thus pretreated liquor and adding thereto a sorbitan ester ethylene oxide as a secondary emulsifier followed by emulsification. The less expensive and stable artificial lipid makes it possible to provide a nonviral carrier whereby a drug can be effectively delivered to an affected part without being captured by phagocytes in vivo. By using a cationic peptide lipid and adding a plasmid gene and a nucleic acid-binding protein thereto, the gene can be effectively immobilized to the lipid vesicles and applied to gene therapy.

Description

明 細 書  Specification
脂質べシクル、 脂質べシクルの製造方法および脂質べシクルへ遺伝子を 固定化する方法 技術分野 TECHNICAL FIELD The present invention relates to a lipid vesicle, a method for producing the lipid vesicle, and a method for immobilizing a gene on the lipid vesicle.
本発明は、 癌治療や遺伝子治療のための D D Sにおける、 薬物キヤリ ァとして有用な脂質べシクル （脂質分子膜からなる閉鎖小胞体） および 脂質べシクルへの遺伝子の固定化方法に関するものである。  The present invention relates to a lipid vesicle (closed endoplasmic reticulum composed of a lipid molecular membrane) useful as a drug carrier and a method for immobilizing a gene to the lipid vesicle in DDS for cancer therapy or gene therapy.
背景技術 Background art
D D Sに使用される非ウィルス性キヤリアとしては、 脂質膜成分の 5 0 %以上 （現実的にはほぼ全て） が天然リ ン脂質よりなるリボソームで あり、 これらは逆相蒸発法あるいは超音波照射法などによって調製され ている。 しかしながらこれらのリボソームは、 ①リボソーム内部への物 質包括率が小さい、 ②リボソームの内水相容積が小さいため、 遺伝子な どのマクロ分子を包括させ難い、 ③リボソームの安定性が悪い、 ④高価 である、 などの問題があった。  In non-viral carriers used in DDS, ribosomes composed of natural phospholipids account for 50% or more (actually almost all) of the lipid membrane components, and these are the reverse-phase evaporation method or ultrasonic irradiation method. It has been prepared. However, these ribosomes have the following problems: (1) the inclusion ratio of substances inside the ribosome is small; (2) the small volume of the internal aqueous phase of the ribosome makes it difficult to include macromolecules such as genes; (3) the stability of the ribosome is poor; There was a problem.
これに対し、 ソノレビタンエステルで人工脂質膜を構成したべシクルの 製法も提案されており、 安定性にすぐれしかも安価なキャリアーとして 期待されている。 ところで癌治療等で化学療法を行う場合に、 薬剤が標 的となる患部の細胞以外の正常細胞をも傷つけてしまうという副作用の 問題がある。 たとえば、 抗癌剤を使用する場合には副作用が伴う場合が あり、 副作用を軽減するよぅ抗癌剤の投与量を抑えれば癌細胞に十分に 作用することはできない。 さらに、 体内にはマクロファージのような食 細胞があり、 キャリア一としてリボソームやべシクルを投与しても患部 に達して作用する前に食細胞に貪食 （捕捉 ·分解） されてしまうという 差替え用紙 （規則 26) 問題がある。 On the other hand, a method for producing a vesicle in which an artificial lipid membrane is composed of sonolebitan ester has been proposed, and is expected to be a stable and inexpensive carrier. By the way, when performing chemotherapy for cancer treatment or the like, there is a problem of a side effect that the drug also damages normal cells other than cells in the target affected area. For example, when an anticancer drug is used, it may have side effects. To reduce the side effects, if the dose of the anticancer drug is reduced, it cannot sufficiently act on cancer cells. In addition, there are phagocytes such as macrophages in the body, and even if ribosomes or vesicles are administered as carriers, they are phagocytosed (captured / degraded) by the phagocytes before reaching the affected area and acting. (Rule 26) There's a problem.
また、 脂質べシクルは外部がマイナス極性であり、 同じくマイナス極 性である D N Aを付着させにくいという問題がある。 従って、 従来の脂 質べシクルでは D N Aの付着効率が悪く、 遺伝子キヤリアとして遺伝子 治療等に使用するのは困難である。  In addition, lipid vesicles have a problem in that the outside has a negative polarity, and it is difficult to attach DNA, which is also a negative polarity. Therefore, conventional lipid vesicles have poor adhesion efficiency of DNA, and it is difficult to use them as gene carriers for gene therapy and the like.
本発明の目的とするところは、 食細胞に捕捉されにく く、 効果的な化 学治療ができる D D Sにおけるキャリアーとして有効な脂質べシクルお よびその製法を提供することにある。 また、 キャリアーとしての脂質べ シクルに目標である細胞に対する標的指向性 （ミサイル機能） を付与す ることも目的とする。 さらに、 脂質べシクル上に遺伝子を効果的に固定 することも目的とする。 発明の開示  An object of the present invention is to provide a lipid vesicle which is not easily captured by phagocytic cells and can be used as a carrier in DDS which enables effective chemical treatment, and a method for producing the same. Another objective is to give the lipid vesicle as a carrier the target directivity (missile function) for the target cell. Another object is to effectively fix genes on lipid vesicles. Disclosure of the invention
本発明に係る脂質べシクルの製造方法は、 一次乳化剤としてソルビタ ンエステルと溶媒を含む処理液に水を加えて、 超音波照射を行って乳化 させる工程と、 前記処理が行われた処理液から溶媒を留去し、 これに二 次乳化剤としてソルビタンエステルのエチレンォキシド付加物を配合し 乳化させる工程を含む二段階乳化法である。 また、 コレステロールと レ シチンを含む処理液の溶剤にへキサンを用い、 これに一次乳化剤として ソルビタンエステルを配合し、 水を加えて、 超音波照射を行って乳化さ せる工程と、 前記処理が行われた処理液から溶媒を留去し、 その残液に 二次乳化剤と してソルビタンエステルのエチレンォキシド付加物を配合 し、 攪拌させて乳化させる工程を含む脂質べシクルの製造方法でもよい, —次乳化剤と して、 ソルビタンォレイン酸モノエステルを用いてもよく 二次乳化剤と して、 ソルビタンォレイン酸モノエステルのエチレンォキ シド付加物を用いてもよい。 一次乳化の際に処理液を冷却しながら超音 波照射を行ってもよい。 さ らに、 メンブランろ過を行い、 粒径が 2 0 0 n m以下のナノサイズ脂質べシクルを通過させてもよい。 The method for producing a lipid vesicle according to the present invention includes: a step of adding water to a treatment liquid containing a sorbitan ester as a primary emulsifier and a solvent, and emulsifying the mixture by ultrasonic irradiation; and This is a two-stage emulsification method that includes the step of distilling off and then emulsifying and emulsifying a secondary emulsifier with an ethylene oxide adduct of a sorbitan ester. Also, a step of using hexane as a solvent of the treatment liquid containing cholesterol and lecithin, blending sorbitan ester as a primary emulsifier with this, adding water and emulsifying by ultrasonic irradiation, and The method of producing a lipid vesicle may include a step of evaporating a solvent from the treated solution, adding a sorbitan ester ethylene oxide adduct as a secondary emulsifier to the remaining solution, and stirring and emulsifying the mixture. —Sorbitan oleic acid monoester may be used as the secondary emulsifier, and sorbitan oleic acid monoester ethylene oxide adduct may be used as the secondary emulsifier. Ultra sound while cooling the processing liquid during primary emulsification Wave irradiation may be performed. Further, the membrane may be subjected to membrane filtration to pass through a nano-sized lipid vesicle having a particle size of 200 nm or less.
本発明に係る別の脂質べシクルの製造方法は、 一次乳化剤と しての ソルビタンエステルを溶解した溶媒に水を加えて処理液と し、 この処理 液を冷却しながら超音波照射を行って乳化させる一次乳化工程と、 前記 処理が行われた処理液から溶媒を留去し、 これに二次乳化剤と してソル ビタンエステルのエチレンォキシド付加物と P E G脂質を配合して乳化 させる二次乳化工程を含むことを特徴とするものである。 前記ソルビタ ンエステルと してソルビタンォレイン酸モノエステルを、 前記溶媒と し てへキサンを用いることが好ましく、 前記 P E G脂質と して脂質誘導体 を持つ P E G脂質を用いることが望ましい。 また、 一次乳化工程におい て処理液にさらにィソチオシアン酸オタタデシルエステル （ I A O E ) を混入するとともに、 二次乳化工程の後に、 可溶性プロテイン Aを混入 する工程と、 抗体を添加する工程を含むことが好ましい。  In another method for producing a lipid vesicle according to the present invention, a treatment liquid is prepared by adding water to a solvent in which a sorbitan ester as a primary emulsifier is dissolved, and the treatment liquid is cooled and subjected to ultrasonic irradiation while being emulsified. A primary emulsification step in which the solvent is distilled off from the treated solution subjected to the above-mentioned treatment, and a secondary emulsifier in which an ethylene oxide adduct of a sorbitan ester and a PEG lipid are blended and emulsified as a secondary emulsifier. It is characterized by including a step. It is preferable to use sorbitan oleic acid monoester as the sorbitan ester and hexane as the solvent, and it is desirable to use a PEG lipid having a lipid derivative as the PEG lipid. In addition, in the primary emulsification step, the process solution may further include a step of mixing iotadecyl ester of isothiocyanic acid (IAOE) into the treatment solution, and a step of mixing soluble protein A after the secondary emulsification step and a step of adding an antibody. preferable.
本発明に係る脂質べシクルに遺伝子を固定化する方法は、 プラスミ ド遺伝子に核酸結合性タンパクを加え、 カチオン性ぺプチド脂質を含む 人工脂質より構成される脂質べシクルと混合することにより遺伝子を固 定化するものである。 核酸結合性タンパクは、 ヒス トン、 プロタミン及 びポリ Lリジンからなる群から選ばれることが好ましく、 さらにそれら を 2つ以上組合わせることが好ましい。  The method for immobilizing a gene on a lipid vesicle according to the present invention comprises the steps of: adding a nucleic acid-binding protein to a plasmid gene; and mixing the gene with a lipid vesicle composed of an artificial lipid containing a cationic peptide lipid. This will be fixed. The nucleic acid binding protein is preferably selected from the group consisting of histone, protamine and poly-L-lysine, and more preferably two or more of them are combined.
本発明に係る脂質べシクルは、 上述の製造方法によ り製造されたも の、 又は上述の遺伝子を固定化する方法により遺伝子を固定化したもの である。 また、 本発明に係る脂質べシクルは、 カチオン性ペプチド脂質 を含む人工脂質と、 プラス ミ ド遺伝子と、 プラスミ ド遺伝子と、 ヒス ト ン、 プロタミン及びポリ L リジンからなる群から選ばれる核酸結合性タ ンパクを有するものでもよい。 本発明に係る別の脂質べシクルは、 ソル Λ The lipid vesicle according to the present invention is one produced by the above-mentioned production method, or one obtained by immobilizing a gene by the above-mentioned method for immobilizing a gene. Further, the lipid vesicle according to the present invention comprises an artificial lipid containing a cationic peptide lipid, a plasmid gene, a plasmid gene, and a nucleic acid-binding agent selected from the group consisting of histamine, protamine and poly-L-lysine. It may have a protein. Another lipid vesicle according to the present invention is sol Λ
4  Four
ビタンエステルで構成される膜と、 当該膜に付着した脂質誘導体を持つ P E G脂質と、 プロテイン Aと結合した抗体と、 前記プロテイン Aを結 合させる脂質アンカーのと してィソチオシアン酸オタタデシルエステル ( I A O E ) を有することを特徴とするものである。 図面の簡単な説明 A membrane composed of a vitan ester, a PEG lipid having a lipid derivative attached to the membrane, an antibody bound to protein A, and otathidecyl isosothiocyanate as a lipid anchor for binding the protein A ( IAOE). BRIEF DESCRIPTION OF THE FIGURES
第 1図は本発明の脂質べシクルの製造における一次乳化の工程を示す 断面図である。 第 2図は全脂質に占めるレシチンとコレステロール割合 とべシクルの生成率の関係を示すグラフである。 第 3図はマイクロサイ ズべシクルの調製における全脂質に占める P E G脂質の割合とべシクル の生成率の関係を示すグラフである。 第 4図はナノサイズべシクルの調 製における全脂質に占める P E G脂質の割合とべシクルの生成率の関係 を示すグラフである。 第 5図は本発明に係る遺伝子を固定化する方法の 説明図である。 第 6図は本発明に係る遺伝子を固定化する方法をマイク 口サイズべシクルに適用した結果を示すグラフである。 第 7図は同ダラ フのさらに詳細なグラフである。 第 8図は本発明に係る遺伝子を固定化 する方法をナノサイズべシクルに適用した結果を示すグラフである。 第 9図はヒス トンを用いて本発明に係る遺伝子を固定化する方法を適用し た結果を示すグラフである。 第 1 0図は核酸結合性タンパクの各種組み 合わせを比較した結果を示すグラフである。 発明を実施するための最良の形態  FIG. 1 is a cross-sectional view showing a step of primary emulsification in the production of a lipid vesicle of the present invention. FIG. 2 is a graph showing the relationship between the ratio of lecithin and cholesterol in total lipids and the rate of vesicle formation. FIG. 3 is a graph showing the relationship between the ratio of PEG lipids to the total lipids in the preparation of microsize vesicles and the production rate of vesicles. FIG. 4 is a graph showing the relationship between the percentage of PEG lipids in the total lipids and the vesicle generation rate in the preparation of nano-sized vesicles. FIG. 5 is an explanatory diagram of a method for immobilizing a gene according to the present invention. FIG. 6 is a graph showing the result of applying the method for immobilizing a gene according to the present invention to a micropore-sized vesicle. Figure 7 is a more detailed graph of the Daraf. FIG. 8 is a graph showing the results of applying the method for immobilizing a gene according to the present invention to nano-sized vesicles. FIG. 9 is a graph showing the result of applying the method of immobilizing a gene according to the present invention using a histone. FIG. 10 is a graph showing the results of comparing various combinations of nucleic acid binding proteins. BEST MODE FOR CARRYING OUT THE INVENTION
本発明をより詳細に示すために、 添付の図面に従ってこれを説明する。 本発明の二段階乳化法によるナノサイズ脂質べシクルの製法の基本的 な実施の形態は、 以下のとおりである。  In order to illustrate the present invention in more detail, it will be described with reference to the accompanying drawings. The basic embodiment of the method for producing a nano-sized lipid vesicle by the two-stage emulsification method of the present invention is as follows.
べシクルの主成分となる ソルビタンォレイン酸モノエステル （商品名 「スパン 8 0 j 和光純薬製） と、 予めへキサン中に溶解させたレシチン とコ レステロールを、 ホモミキサーによって攪拌しながら少しずつ水を 滴下し、 超音波ホモジナイザーを用いて超音波照射を繰り返し、 前記 W ZO型エマルションをフラスコに入れ減圧下でへキサンを除去し、 この よ う にして調製された W/L型エマルシヨ ン中に、 ソルビタンォレイ ン 酸モノエステルのエチレンォキシド付加物 （商品名 「ツイーン 8 0」 和 光純薬製） の水溶液を攪拌混合したのち、 遠心分離すれば良い。 以下、 実施例により、 さらに詳細に説明する。 Sorbitan oleate monoester (trade name) “Span 80 j manufactured by Wako Pure Chemical Industries, Ltd.) and lecithin and cholesterol previously dissolved in hexane were added dropwise while stirring with a homomixer, and ultrasonic irradiation was repeated using an ultrasonic homogenizer. Then, the WZO-type emulsion was placed in a flask, and hexane was removed under reduced pressure. The thus-prepared W / L-type emulsion contained ethylene oxide adduct of sorbitan oleic acid monoester ( An aqueous solution of “Tween 80” (product of Wako Pure Chemical Industries) may be stirred and mixed, and then centrifuged. Hereinafter, the present invention will be described in more detail with reference to examples.
第 1図は本実施例の一次乳化の工程を示す断面図である。 試験管、 ビ 一力等の内容器 1にべシクルの主成分となるソルビタンォレイン酸モノ エステル （商品名 「スパン 8 0 J 和光純薬製） 6 6 m gと、 予め n—へ キサン 1 m l 中に 5 m g となるように溶解させておいたレシチン、 コレ ステロールを必要な量 （ 6 m g , 3 m g ) と り、 全量が 3 m l となるよ うに n—へキサンを加えた処理液 2を入れ、 ホモミキサー （ 1 5 , 0 0 0 r p m) で 3 0秒間攪拌する。 その溶液をホモミキサーによって攪拌 しながら、 包括物質について所定の濃度に調製した内水相となる水溶液 3 (0. 1 5 m l ) を少しずつ滴下し （ 1分間）、 その後、 超音波ホモジ ナイザー 4を用いて超音波照射を行う。 この超音波照射は、 1 5秒行つ て 1 5秒休むというサイクルを 3回繰り返した。 內容器 1の外側に外容 器 5を設け、 内容器 1 と外容器 5の間に氷水 6を入れて処理液 2を冷却 することにより、 超音波照射による処理液 2の温度上昇を抑え、 処理液 2中の n—へキサンが発火することを防止している。 超音波ホモジナイ ザ一 4の出力は、 内容器 1に入れる処理液 2の量や目標とするべシクル の粒径によって適宜選択するが、 試験管程度の小規模な容器で 2 0 0 n m以下のナノサイズを得るには、 5 0〜 1 4 0 W程度のものが適してお り、 特に 1 0 0 n m以下の粒径を得るには 8 0〜 1 4 0 W程度のものが _ FIG. 1 is a cross-sectional view showing a primary emulsification process of this example. Sorbitanoleic acid monoester (trade name “Span 80 J made by Wako Pure Chemical Industries, Ltd.”), which is the main component of the vesicle, in a test tube, inner container such as a test vessel, etc. 66 mg, and n-hexane 1 ml in advance Take Lecithin and Cholesterol, which were dissolved to a concentration of 5 mg, in the required amount (6 mg, 3 mg), and add treated solution 2 to which n-hexane was added so that the total amount was 3 ml. The mixture is stirred for 30 seconds with a homomixer (15, 000 rpm), and while stirring the solution with a homomixer, an aqueous solution 3 (0.1. 5 ml) was added dropwise little by little (1 minute), and then ultrasonic irradiation was performed using an ultrasonic homogenizer 4. This ultrasonic irradiation was repeated three times, a cycle of 15 seconds and a rest of 15 seconds.設 け Install outer container 5 outside container 1 and place ice between inner container 1 and outer container 5. Cooling the processing solution 2 by adding 6 suppresses the temperature rise of the processing solution 2 due to the ultrasonic irradiation and prevents the n-hexane in the processing solution 2 from igniting. The output of (4) is appropriately selected depending on the amount of the processing solution 2 to be put into the inner container 1 and the target particle diameter of the vesicle, but it is necessary to obtain a nano size of 200 nm or less in a small vessel such as a test tube. Is suitable for a particle size of about 50 to 140 W, especially for a particle size of 100 nm or less. _
6  6
適している。 このようにして得られた wz o型エマルションをナスフラ スコに入れ、 ロータリ一エバポレーターで滅圧させて n—へキサンを除 去し、 このようにして得た W/L型エマルシヨン中に、 ソルビタンォレ イン酸モノエステルのエチレンォキシ ド付加物水溶液 [商品名 「ツイ一 ン 8 0」 （ l S m g Zm l ) 和光純薬製] 3 m l を入れ、 ホモミキサー ( 3 0 0 0 r p m) を用いて攪拌したのち、 遠心分離をして行ってナノ サイズ脂質べシクルを調製した。 前記ナノべシクルの大きさは、 透過型 電子顕微鏡によって観察を行い、 その結果前記べシクルの平均粒径は 4 0〜1 0 0 n mであることが確認された。 Are suitable. The wzo emulsion obtained in this manner is put into a NASA flask, depressurized by a rotary evaporator to remove n-hexane, and sorbitan oil is added to the W / L emulsion thus obtained. 3 ml of an aqueous solution of an ethylene oxide adduct of an acid monoester [trade name “Twin 80” (l S mg Zm l) manufactured by Wako Pure Chemical Industries) was added, and the mixture was stirred using a homomixer (3000 rpm). Then, centrifugation was performed to prepare nano-sized lipid vesicles. The size of the nano vesicle was observed by a transmission electron microscope, and as a result, it was confirmed that the average particle size of the vesicle was 40 to 100 nm.
このようにして得られた脂質べシクルはそれ自体が新規なものである < すなわち、 従来のリボソームやマイク口サイズの脂質べシクルと異なり . サイズは 1 0 0 n m程度又はそれ以下と小さく、 さらにその内部に物質 を高い包括率で内包させることができる構造となっている。 サイズは比 較的均一であり、 超音波照射の周波数、 強度、 時間を変えることによつ てある程度調整できる。 さらにメ ンブランろ過を行うことにより、 滅菌 処理とともにそのサイズを 2 0 0 n m以下のいわゆるナノサイズに （特 に、 超音波を十分に照射した場合は l O O n m以下に） そろえることが できる。 生理丧塩水中での安定性は良好であり、 血清中においても投入 時に 3 0 %程度の損傷がある他は安定に存在し続けている。 さらに、 リ ンパ球にも良好に接触し、 内包物質を機能させることができた。  The lipid vesicles obtained in this manner are novel in themselves. That is, unlike conventional ribosomes and microphone-sized lipid vesicles, the size is as small as about 100 nm or less. It has a structure in which substances can be contained at a high inclusion rate. The size is relatively uniform and can be adjusted to some extent by changing the frequency, intensity, and duration of the ultrasonic irradiation. Furthermore, by performing membrane filtration, the size can be adjusted to the so-called nano size of 200 nm or less (especially, to less than 100 nm when sufficiently irradiated with ultrasonic waves) together with the sterilization treatment. The stability in physiological saline is good, and it remains stable even in serum except for about 30% of damage when injected. In addition, it was in good contact with the lymphocytes and the encapsulated substance was able to function.
前記実施例において、 ハイプリ ッ ド型ナノべシクルの生成率を調べる ため、 ソノレビタンォレイ ン酸モノエステル、 レシチン及びコレステロ一 ルの比率を変化させて、 その最適条件を調べた。 トレーサーとして、 0. 1 %イ ンジゴカルミ ン 0. 1 5 m 1 を用いて、 前記と同様の二段階乳化 法によって調製し、 遠心分離、 ゲルフィルトレーシヨンを行ったのち、 分光光度計を用いて 6 2 0 n mでべシクル溶液 0. 5 m 1 に 1 0 % S D S (物質名 ドデシル硫酸ナトリ ウム） 0. 5 m 1 を加え良く攪拌して吸 光度を測定した。 In the above example, in order to examine the generation rate of hybrid type nanovesicles, the optimal conditions were examined by changing the ratio of sonolebitanoleate monoester, lecithin and cholesterol. Prepare 0.1% indigo carmine 0.15m1 as a tracer by the same two-stage emulsification method as above, centrifuge and gel filtration, and then use a spectrophotometer. Vesicle solution at 62 nm 0.5% SD in 10% SD 0.5 ml of S (substance name sodium dodecyl sulfate) was added, and the mixture was stirred well and the absorbance was measured.
これらの試験結果は第 2図に.示したとおりであり、 膜強化剤であるレ シチンとコレステロールが含まれていない場合の生成率は約 4 0 %とな つており、 レシチンと コ レステロールの両者を併用することによって、 生成率の最大値は約 7 0 %となっており、 その配合比はソルビタンォレ イン酸モノエステノレ 1 3 2 m g、 レシチン 1 2 m g、 コ レステロ一ノレ 6 m gであった。 レシチンと コ レステロ一ルを全脂質に対して 3〜 1 3 % に調整することにより、 生成率を 6 0 %以上に保つことができる。  The results of these tests are shown in Figure 2, where the production rate without the membrane enhancers lecithin and cholesterol was about 40%, indicating that both lecithin and cholesterol were present. The maximum value of the production rate was about 70% by the combined use, and the compounding ratio was 132 mg of monoestenol sorbitanoleate, 12 mg of lecithin, and 6 mg of cholesterol monol. By adjusting lecithin and cholesterol to 3 to 13% of total lipids, the production rate can be maintained at 60% or more.
前記ナノべシクルの大きさは、 透過型電子顕微鏡によって観察を行い, その結果前記べシクルの平均粒径は 4 0〜 1 0 0 n mであることが確認 された。  The size of the nano vesicle was observed by a transmission electron microscope, and as a result, it was confirmed that the average particle size of the vesicle was 40 to 100 nm.
次に別の実施例を示す。 ソルビタンォレイン酸モノエステル （商品名 「スノヽ。ン 8 0」） 1 3 2 m gにレシチン 6. O Om g と コ レステロ一ノレ 1 2 , O m gを添加し、 n—へキサン 4. 0 0 m l に溶解させて、 マイク 口ホモジナイザ一で 3 0秒間攪拌した。 次いで内水相となる包括物質の 水溶液を 0. 3 0 m l滴下しながら 1分間攪拌したのち、 超音波ホモジ ナイザーによる超音波照射を行い、 一次乳化を行った。 前記処理液をナ スフラスコに入れ、 ロータリ一エバポレータ一で減圧して n—へキサン を除去した。 ここでソルビタンォレイ ン酸モノエステルのエチレンォキ シド付加物水溶液 （商品名 「ツイーン 8 0」） 4 2. O m l を生理食塩水 4. 0 0 m 〗 に溶解させた水溶液 （P E G脂質べシクルの調製の場合は. ここに P E G脂質を必要量添加しておく） をナスフラスコに混ぜ、 大き な wzozw型エマルシヨンの脂質の塊がなくなるく らいまで、 マイク 口ホモジナイザーで攪拌し二次乳化を行った。 その溶液をビーカに入れ. ホモミキサー （ 3 0 0 0 r p m) を用いて攪拌したのち、 遠心分離をし g Next, another embodiment will be described. Sorbitanoleic acid monoester (trade name “Snod. 80”) Add 13 mg of lecithin to 12 mg of cholesterol and 12 mg of cholesterol mono-oleate, and add 0.1 mg of n-hexane to 4.0 mg of n-hexane. The mixture was dissolved in 0 ml and stirred with a microphone-mouth homogenizer for 30 seconds. Next, the mixture was stirred for 1 minute while adding 0.30 ml of an aqueous solution of the inclusion substance to be an internal aqueous phase, and then subjected to ultrasonic irradiation with an ultrasonic homogenizer to perform primary emulsification. The treatment liquid was put in a NAS flask, and the pressure was reduced by a rotary evaporator to remove n- hexane. Here, an aqueous solution of ethylene oxide adduct of sorbitan oleic acid monoester (trade name “Tween 80”) 42. An aqueous solution obtained by dissolving O ml in 4.0 ml of physiological saline (PEG lipid vesicle In the case of preparation. Add the required amount of PEG lipid here) to an eggplant flask, and perform secondary emulsification by stirring with a microphone-mouth homogenizer until the large mass of wzozw-type emulsion lipids disappears. . Place the solution in a beaker. Stir using a homomixer (3000 rpm), then centrifuge. g
て行ってナノサイズ脂質べシクルを調製した。 本発明に係る脂質べシク ルの製造方法は、 大小さまざまな規模で適用できるが、 特に比較的小規 模で簡易に実施するためには、 ソルビタンエステル 1 0〜 1 O O O m g , ソノレビタンエステノレのェチレンォキシド付カ [J物 1 0〜 5 0 m g、 べシク ルの内水容量 0. 1〜 1 m 1、 外水容量 1〜 1 0 m 1 の範囲に調整する のがよい。 To prepare a nano-sized lipid vesicle. Although the method for producing lipid vesicles according to the present invention can be applied on various scales, large and small, in particular, for relatively small and simple implementation, sorbitan esters 10 to 1 OOO mg, sonorebitan ester It is advisable to adjust the volume of water with ethylenoxide to the range of 10 to 50 mg, the internal water volume of the vesicles to 0.1 to 1 m1, and the external water volume to 1 to 10 m1.
前記ナノべシクルの大きさは、 透過型電子顕微鏡によって観察を行い、 その結果前記べシクルの平均粒径は約 40〜 1 0 0 n mであることを確 認した。  The size of the nano vesicle was observed by a transmission electron microscope, and as a result, it was confirmed that the average particle diameter of the vesicle was about 40 to 100 nm.
本発明に係る脂質べシクルの製造方法を、 体内の食細胞による貪食を 回避することができる機能 （ステルス機能） を付加したべシクルの製造 に適用する実施の形態について説明する。 べシクルの主成分となるソル ビタンォレイン酸モノエステルと、 予めへキサン中に溶解させたレシチ ンとコ レステロールを、 ホモミキサ一によって攪拌しながら少しずつ水 を滴下し、 超音波ホモジナイザーを用いて超音波照射を繰り返し （一次 乳化工程） 前記 wZo型エマルシヨ ンを得る。 この w/o型エマルショ ンをフラスコに入れ減圧下でへキサンを除去し、 このようにして調製さ れた W/L型エマノレショ ン中に、 ソルビタンォレイ ン酸モノエステ/レの エチレンォキシド付加物と脂質誘導体を持つ P E G脂質 （商品名 「SU NB R I GHT D S P E - 2 0 HC J 日本油脂株式会社製） の水溶 液を攪拌混合したのち （2次轧化工程）、 遠心分離すれば良い。 粒径が 1 μ m程度のマイクロサイズ脂質べシクルを調製する場合は、 一次乳化工 程において超音波照射の代わりにホモミキサ一によつて攪拌すればよい c 以下、 実施例によりステルス機能を持つ脂質べシクルの製造方法につい てさらに詳細に説明する。 An embodiment will be described in which the method for producing a lipid vesicle according to the present invention is applied to the production of a vesicle to which a function (stealth function) capable of avoiding phagocytosis by phagocytes in the body is added. Sorbitanoleic acid monoester, which is the main component of the vesicle, and lecithin and cholesterol previously dissolved in hexane are added dropwise little by little with stirring using a homomixer, and ultrasonic waves are applied using an ultrasonic homogenizer. The irradiation is repeated (primary emulsification step) to obtain the wZo-type emulsion. The w / o emulsion was placed in a flask to remove hexane under reduced pressure, and the ethylene oxide of sorbitan monooleate monoester was added to the W / L emulsion thus prepared. An aqueous solution of a PEG lipid having an adduct and a lipid derivative (trade name “SU NB RI GHT DSPE-20 HC J Nippon Oil & Fats Co., Ltd.”) may be mixed by stirring (secondary conversion step) and then centrifuged. . If the particle size to prepare a micro-sized lipid base Shikuru about 1 mu m, the primary in emulsion Engineering degree may be due connexion stirring homomixer one instead of ultrasonic irradiation c below, a stealth by example The method for producing a lipid vesicle will be described in more detail.
本実施例の一次乳化の工程を第 1図に示す。 試験管、 ビーカ等の内容 Ω FIG. 1 shows the primary emulsification process of this example. Contents of test tubes, beakers, etc. Ω
y  y
器 1にべシクルの主成分となるソルビタンォレイン酸モノエステル （商 品名 「スパン 8 0」 和光純薬製） およびレシチンとコ レステロールを入 れ、 n—へキサン （ 4 m l ) に溶解させる。 この溶液をホモミキサー ( 1 5， O O O r p m) で 3 0秒間攪拌する。 べシクル内の包括物質を 所定の濃度に調製した内水相となる水溶液 3 (0. 3 m l ) を少しずつ 滴下しながら 1分間ホモミキサーによって攪拌しながら、 その後、 超音 波ホモジナイザー 4を用いて超音波照射を行う。 この超音波照射は、 1 5秒行って 1 5秒休むというサイクルを 3回繰り返した。 内容器 1の外 側に外容器 5を設け、 内容器 1 と外容器 5の間に氷水 6を入れて処理液 2を冷却することにより、 超音波照射による処理液 2の温度上昇を抑え、 処理液 2中の n—へキサンが発火することを防止している。 このように して得られた WZO型エマルショ ンをナスフラスコに入れ、 ロータ リー エバポレーターで減圧させて 2 8でで n—へキサンを除去する。 そして, ナスフラスコの側面についた物質を割り箸等で作ったヘラで削り落とす このようにして得た WZL型エマルシヨン中に、 ソルビタンォレイン酸 モノエステルのエチレンォキシド付加物水溶液と P E G脂質を入れ、 ホ モミキサー （ 3 000 r p m) を用いて攪拌したのち、 遠心分離を行つ てナノサイズ脂質べシクルを調製した。 Put sorbitan oleate monoester (trade name “Span 80”, manufactured by Wako Pure Chemical Industries, Ltd.) and lecithin and cholesterol, which are the main components of the vesicle, into vessel 1 and dissolve in n-hexane (4 ml). This solution is stirred for 30 seconds with a homomixer (15, O O O rpm). Aqueous solution 3 (0.3 ml), which becomes the internal aqueous phase in which the inclusion substance in the vesicle was adjusted to a predetermined concentration, was added dropwise little by little while stirring with a homomixer for 1 minute, and then using an ultrasonic homogenizer 4. To irradiate ultrasonic waves. This ultrasonic irradiation was repeated for 15 seconds and then rested for 15 seconds three times. An outer container 5 is provided outside the inner container 1, and ice water 6 is put between the inner container 1 and the outer container 5 to cool the processing liquid 2, thereby suppressing the temperature rise of the processing liquid 2 due to ultrasonic irradiation. It prevents the n-hexane in the treatment liquid 2 from igniting. The WZO emulsion thus obtained is placed in an eggplant-shaped flask, and the pressure is reduced by a rotary evaporator, and n-hexane is removed at 28. Then, the substance attached to the side of the eggplant flask is scraped off with a spatula made with chopsticks etc.Into the WZL emulsion obtained in this way, an aqueous solution of ethylene oxide adduct of sorbitan oleic acid monoester and PEG lipids are added. After stirring using a homomixer (3,000 rpm), the mixture was centrifuged to prepare nano-sized lipid vesicles.
以上、 ナノサイズべシクルの調製を例に説明したが、 一次乳化の際に 超音波照射を行わずホモミキサーのみで撹拌することにより、 上記製法 はマイクロサイズべシクルの調製にも適用することができる。  As described above, the preparation of nano-sized vesicles has been described as an example.However, the above method can be applied to the preparation of micro-sized vesicles by stirring only with a homomixer without performing ultrasonic irradiation during primary emulsification. it can.
上述の通り、 べシクルの主成分であるソルビタンォレイン酸モノエス テルと して市販の製品である商品名 「スパン 8 0 J を使用した。 ソルビ タンォレイ ン酸モノエステルは 1本の脂肪酸の鎖を持つ構造であるが、 市販されている製品には 2本又はそれ以上の鎖を持つ不純物が含まれて いる。 そして、 この不純物を含む製品を用いる方が純粋なソルビタンォ 丄 0 As described above, Sorbitan oleate monoester, a commercial product, was used as sorbitan oleate monoester, which is the main component of the vesicle. The sorbitan oleate monoester has a single fatty acid chain. Although commercially available products contain impurities with two or more chains, it is better to use products containing these impurities than pure sorbitan. 丄 0
レイ ン酸モノエステルを用いるよ り も良好にべシクルを生成できること 力 わ力、つた。 The ability to produce vesicles better than using the monoester of leic acid.
また、 本例の製法において加える P E G脂質のスパン 8 0に対する最 適な比率も明らかになった。 この結果を第 3図および第 4図に示す。 第 3図はマイクロサイズべシクルの場合であり、 第 4図はナノサイズべシ クルの場合である。 横軸にはスパン 8 0に対する P E G脂質のモル％で ある。 一方、 縦軸はべシクルの生成率であり、 べシクルの一次乳化調製 時に滴下した水溶液に含ませておいた蛍光物質カルボキシフルォレセィ ン （C F ) の濃度と最終的に生成したべシクルの内層水超に中に取り込 まれた C Fの濃度の比率で示した数値である。 これより、 スパン 8 0に 対して P E G脂質が 2 m o 1 %程度のときが生成率が高く、 最適値であ ることがわかる。  In addition, the optimal ratio of PEG lipid added to span 80 in the production method of this example was also clarified. The results are shown in FIGS. 3 and 4. Fig. 3 shows the case of micro-sized vesicles, and Fig. 4 shows the case of nano-sized vesicles. The abscissa is the mole percentage of PEG lipid per span 80. On the other hand, the vertical axis represents the vesicle formation rate. The concentration of the fluorescent substance carboxyfluorescein (CF) contained in the aqueous solution dropped during the primary emulsification preparation of the vesicle and the final vesicle formation It is a numerical value expressed as the ratio of the concentration of CF taken into the water above the inner layer. From this, it can be seen that the generation rate is high and the optimum value is obtained when the PEG lipid content is about 2 mol% for span 80.
また本例の製法によって生成した P E G脂質べシクルと P E Gを含ま ないべシ ルを生理的食塩水中に 3 0 日おいて安定性を比較した。 マイ クロサイズべシクル、 ナノサイズべシクルともに P E G脂質べシクルの 方が安定性が高いことが確認された。  In addition, the stability of PEG lipid vesicles produced by the method of this example and vesicles without PEG were compared for 30 days in physiological saline. It was confirmed that both the micro- and nano-sized vesicles had higher stability with the PEG lipid vesicle.
以上、 表面に P E G脂質を持つ親水性の高い脂質べシクルの製法につ いて述べた。 本例の製法によって生成した P E G脂質べシクルを食細胞 の一種であるマクロファージの U 9 3 7細胞と接触させたところ、 P E G脂質を含まないべシクルに比べて U 9 3 7細胞に貪食されにくいこと が観察され、 食細胞からの回避機能 （ステルス機能） を備えることが確 認された。 マイクロサイズべシクル、 ナノサイズべシクルともにステル ス機能が得られたが、 マイク口サイズべシクルにおいて特に P E G脂質 よるステルス機能向上の効果が高い。 また、 接触させた U 9 3 7細胞の 死滅は P E G脂質べシクルの場合の方が P E G脂質を含まないべシクル より少なく、 細胞毒性が低いことも観察された。 -, -, The method for producing a highly hydrophilic lipid vesicle having a PEG lipid on its surface has been described above. When PEG lipid vesicles produced by the method of this example were brought into contact with U937 cells of macrophages, a type of phagocyte, they were less phagocytosed by U937 cells than vesicles without PEG lipids. Was observed, and it was confirmed to have a function of avoiding phagocytes (stealth function). Stealth function was obtained for both micro-size and nano-size vesicles, but the effect of improving the stealth function with PEG lipids is particularly high for microphone-size vesicles. It was also observed that the killed U933 cells were less with the PEG lipid vesicles than with the PEG lipid-free vesicles and less cytotoxic. -,-,
本実施例において、 ステルス機能を有する P E G脂質べシクルに抗体 を固定化して標的指向性を付与する製法について説明する。 本例の製法 も実施例 1の製法を基本とするが、 さらに抗体を固定するために、 プロ ティ ン Aを固定化する脂質アンカーである I AO Eと抗体を姿勢制御す るためのプロテイン Aおよび抗体を添加する作業が加わる。 In this example, a method for immobilizing an antibody on a PEG lipid vesicle having a stealth function to impart target directivity will be described. The production method of this example is also based on the production method of Example 1, but in order to further immobilize the antibody, I AOE which is a lipid anchor for immobilizing protein A and protein A for controlling the posture of the antibody And an operation of adding an antibody.
べシクル調製に際して、 脂質アンカーであるィソチオシアン酸ォクタ デシルエステル （ I AO E) が必要となるが、 その合成方法について説 明する。 なお、 I AO Eの化学式は C H3 (C H 2) 1 7N C Sである。 ① 1ーァミノォクダデカン （ 1-aminooctadecane) 4. 3 gを 2 5 0 m l のジェチルエーテル （Et20) に溶解する （これを A液とする）。 ② 1， 3—ジシクロへキシルカルボジイ ミ ド （D C C) 3. 4 gを 2 0 0 m l の Et20 に溶かし、 氷と塩で一 1 0°Cに冷却して二硫化炭素 （C S 2) を 8 m l加える （これを B液とする）。 ③ A液を B液にゆっく り加えていく c ④常温で 5時間静置し、 析出したチォ尿素を濾去する。 ⑤濾液を温めな がらエバポレーターにかけて Et20 を取り除く。 ⑥エバポレーターによ る減圧の途中で析出するチォ尿素は、 さらに濾過して取り除く。 ⑦ Et20 がなくなるとオイル状の I AO Eが得られ、 放置すると結晶化する。 ⑧ エタノールで再結晶し精製する。 なお、 反応式は以下の通りである。 In preparing vesicles, octadecyl isothiocyanate (IAOE), which is a lipid anchor, is required. The synthesis method is described below. The chemical formula of IAOE is CH 3 (CH 2 ) 17 NCS. ① Dissolve 4.3 g of 1-aminooctadecane in 250 ml of getyl ether (Et 20 ) (this is referred to as solution A). ② 1, 3- dicyclohexyl to Kishirukarubojii mi de (DCC) 3. Dissolve 4 g in Et 2 0 in 2 0 0 ml, in ice and salt is cooled to a 1 0 ° C and carbon disulfide (CS 2) Add 8 ml (this is B solution). ③ Slowly add solution A to solution B c c Leave at room temperature for 5 hours and filter out the precipitated thiourea. ⑤ filtrate over the evaporator reluctant Na allowed to warm remove the Et 2 0. (4) Thiourea, which precipitates during decompression by the evaporator, is further removed by filtration.オ イ ル Oil-like IAOE is obtained when Et 20 is depleted, and crystallizes when left untreated.再 Recrystallize and refine with ethanol. The reaction formula is as follows.
C S 2 + D C C + CH3 (C H2) 1 7NH2→C H3 (CH2) 1 7N C S +
Figure imgf000013_0001
CS 2 + DCC + CH 3 (CH 2 ) 17 NH 2 → CH 3 (CH 2 ) 17 NCS +
Figure imgf000013_0001
次に、 抗体を固定したべシクルの調製について説明する。 なお、 ここ ではナノサイズべシクルの調製を例に説明するが、 一次乳化の際に超音 波照射を行わずホモミキサ一のみで撹拌することにより、 マイクロサイ ズべシクルの調製にも適用できる。 ①べシクルの主成分となるスパン 8 0 ( 4 3. 7 m g ) に脂質アンカーである I AO E ( 7. 5 m g ： 全脂 質量に対し 1 0 w%) を混入し、 レシチン （0. 8 6 5 m g)、 コレステ χ 2 Next, preparation of a vesicle to which an antibody is immobilized will be described. Here, the preparation of nano-sized vesicles will be described as an example, but the present invention can also be applied to the preparation of micro-sized vesicles by stirring with only a homomixer without performing ultrasonic irradiation during primary emulsification. (1) The lipid anchor I AOE (7.5 mg: 10% by weight based on the total fat mass) is mixed with span 80 (43.7 mg), which is the main component of the vesicle, and lecithin (0. 8 6 5 mg), cholester χ 2
ロール （ 1. 7 3 m g ) を添加して、 試験管を用いて n—へキサン （ 2 m 1 ) に溶解し、 これをホモミキサーで 3 0秒撹拌した後、 撹拌しなが ら包括物質を所定の濃度に調製した水溶液を 0. S m l Zm i nの滴下 速度で 1分間滴下する。 ②試験管を氷水で冷却しながら、 超音波照射を 1 5秒照射 ' 1 5秒停止のサイクルで 3回行う。 ③このようにして得ら れた wZo型エマ^^ショ ンをナスフラスコに入れ、 ロータ リーエバポレ 一ターで減圧させて 2 8°Cで n—へキサンを除去する。 そして、 ナスフ ラスコの側面についた物質を割り箸等で作ったヘラで削り落とす。 ④得 られた L型エマルシヨン中に、 ツイ一ン 8 0 ( 2 1 m g ) と P E G 脂質 「SUN B R I GHT D S P E - 2 0 HCNJ ( 5. 7 5 m g、 す なわちツイーン 8 0と I AO Eの合計量に対し 2 m o l %) を入れ、 ホ モミキサーを用いて攪拌する。 ⑤得られたべシクル溶液に可溶性プロテ イン A ( 5 0 m l ) を混入し、 冷蔵庫に入れて 1 日保管する。 ⑥翌日、 得られた液をマグネチックスターラーで撹拌する。 ⑦べシクル溶液を超 遠心分離して上澄み液を取り除き、 残った液の最も濁度の濃い部分をゲ ル濾過する。 ⑧この溶液に抗ヒ ト I g M抗体を調製した溶液を添加し、 ゆつく り撹拌する。 ⑨再び超遠心分離とゲル濾過を行い抗体を固定化し た P E G脂質べシクルを得る。 Roll (1.73 mg) was added, dissolved in n-hexane (2 ml) using a test tube, and the mixture was stirred for 30 seconds with a homomixer. An aqueous solution prepared to a predetermined concentration is dropped at a dropping rate of 0.1 S ml Zmin for 1 minute. (2) While cooling the test tube with ice water, perform ultrasonic irradiation three times in a cycle of irradiation for 15 seconds and stopping for 15 seconds. (3) Put the obtained wZo-type emulsion into an eggplant flask and reduce the pressure with a rotary evaporator at 28 ° C to remove n-hexane. Then, the substance on the side of the NASFRASCO is shaved off with a spatula made of chopsticks. L Tween 80 (21 mg) and PEG lipid “SUN BRI GHT DSPE-20 HCNJ (5.75 mg, that is, Tween 80 and IAOE) were added to the obtained L-type emulsion. 2 Add 2 mol% to the total amount and stir using a homomixer ⑤ Mix soluble protein A (50 ml) into the obtained vesicle solution and store in a refrigerator for 1 day.液 Stir the obtained solution with a magnetic stirrer 超 Ultracentrifuge the vesicle solution to remove the supernatant, and filter the remaining liquid with the highest turbidity by gel filtration. G. Add the solution containing the IgM antibody and mix gently 撹 拌 Perform ultracentrifugation and gel filtration again to obtain a PEG lipid vesicle on which the antibody is immobilized.
本実施例の製法により調製されたべシクルにおいて、 抗体は抗原結合 点と反対側の部位 （F c部） でプロテイン Aと結合し、 このプロテイン Aは脂質アンカーである I AO Eによって P E G脂質べシクル表面に強 固に固定化されている。 従来の抗体固定化法では P E G脂質でコーティ ングされた脂質べシクルの表面に抗体を姿勢制御して固定化することは 困難であるが、 本例の製法に拠れば抗体の抗原と結合する F a bフラグ メ ン トを外向きにして姿勢制御した形で固定化できる。 また、 P E G脂 質とプロティン Aはそれだけでも両者の親水性親和力によって結合性が l 3 In the vesicle prepared by the production method of this example, the antibody binds to protein A at a site (Fc portion) opposite to the antigen binding point, and this protein A is bound to the PEG lipid vesicle by the lipid anchor I AOE. It is firmly fixed on the surface. With the conventional antibody immobilization method, it is difficult to control and immobilize the antibody on the surface of lipid vesicles coated with PEG lipid, but according to the production method of this example, F The ab fragment can be fixed outward with the attitude controlled. In addition, PEG lipids and protein A alone are capable of binding due to their hydrophilic affinity. l 3
よく、 プロテイン Aによる姿勢制御を P E G脂質べシクルに適用するこ とは極めて有効である。 Often, it is extremely effective to apply protein A posture control to PEG lipid vesicles.
本実施例の製法を用いてマイク口サイズべシクルおよびナノサイズべ シクルを調製し、 癌細胞との接触を行った。 使用した細胞は肺癌患者由 来のリンパ腫と リンパ球とをかけ合わせたハイブリ ドーマである H B 4 C 5細胞である。 H B 4 C 5細胞は抗ヒ ト I g M抗体に対する抗原であ る I g M抗体をその細胞表面に産生する。 脂質べシクルはその内水中に D N A— P Iが含まれるよう調製されており、 接触後に D N A— P I の 作用によって発生する蛍光を観測することにより、 脂質べシクル内の包 括物質 D N A - P Iが H B 4 C 5細胞中に取り込まれたか否かを判定で きるようにしてある。 その結果、 本製法により抗体を固定化じたべシク ルを接触させた場合には P Iの蛍光が H B 4 C 5細胞中に見られた。 一 方、 比較と して抗体を持たないべシクルを接触させた場合には、 P Iの 蛍光が見られなかった。 これらのことより、 本製法によるべシクルは標 的指向性を有することが確認できた。 また、 マイクロサイズべシクルで もナノサイズべシクルでも同様に標的指向性が確認され、 ステルス機能 と標的指向性の両方を同時に備える脂質べシクルの実現に成功した。 なお、 本実施例に係るステルス機能と標的指向性の両方を同時に備え る脂質べシクルは動物実験においても良好な結果を示した。 海藻由来の レクチンの一種である E S Aを固定化したべシクルを用いた。 ここで、 E S Aは癌細胞の D N Aに作用し、 癌細胞のプログラム死、 すなわち、 アポトーシスを誘導させる効果を有する物質である。 初めに、 E S Aを 固定化した脂質べシクルを入れた容器中で培養した癌細胞を実験用ラッ ト 5匹の体に移植する試験を行ったところ、 いずれのラッ トにも癌の発 生がなかったのに対し、 E S Aを固定化していない脂質べシクルを入れ た容器中で培養した癌細胞を実験用ラッ トの体に移植したところ、 5匹 . Microphone-sized vesicles and nano-sized vesicles were prepared using the production method of this example, and were brought into contact with cancer cells. The cells used were HB4C5 cells, which are hybridomas of lymphoma and lymphocytes derived from a lung cancer patient. HB4C5 cells produce IgM antibodies on their cell surface that are antigens for anti-human IgM antibodies. The lipid vesicles are prepared so that DNA-PI is contained in the water, and by observing the fluorescence generated by the action of DNA-PI after contact, the inclusion substance DNA-PI in the lipid vesicles is converted to HB It is possible to determine whether or not it has been taken into 4 C 5 cells. As a result, when the antibody-immobilized vesicle was contacted by this method, PI fluorescence was observed in HB4C5 cells. On the other hand, as a comparison, when the vesicles having no antibody were brought into contact, no PI fluorescence was observed. From these results, it was confirmed that the vesicles produced by this method had the target directivity. In addition, target directivity was confirmed for both micro-sized and nano-sized vesicles, and we succeeded in realizing lipid vesicles that have both stealth function and target directivity. The lipid vesicle according to the present example, which has both the stealth function and the target directivity, showed good results in animal experiments. Vesicles immobilized with ESA, a kind of seaweed-derived lectin, were used. Here, ESA is a substance that acts on DNA of cancer cells and has an effect of inducing programmed death of cancer cells, that is, apoptosis. Initially, a test was conducted in which cancer cells cultured in a vessel containing lipid vesicles with ESA immobilized were transplanted into the body of five experimental rats. In contrast, when cancer cells cultured in a vessel containing lipid vesicles without immobilized ESA were transplanted to the body of an experimental rat, 5 .
14  14
のラッ トの全てに癌が発生した。 これより、 本実施例の脂質べシクルがAll of the rats had cancer. Thus, the lipid vesicles of this example
E S Αを癌細胞に有効に作用させていることが確認された。 さらに、 穂 実施例の脂質べシクルをラ ッ トの体内に注射する試験も行った。 癌細胞 をラッ トの体内に移植した後に脂質べシクルを注射し、 その後の癌細胞 の増殖速度を調査した。 まず、 E S Aを固定化していない脂質べシクル を注射した場合でも、 何も注射しないに場合に比べて癌細胞の増殖を抑 える効果があることがわかった。 さらに、 E S Aを固定化した脂質べシ クルを注射した場合には、 癌細胞の増殖速度が E S Aを固定化していな い脂質べシクルを注射した場合の半分になることも確認された。 It was confirmed that E S Α effectively acted on cancer cells. In addition, a test was conducted in which the lipid vesicles of the Examples were injected into the body of rats. After transplanting the cancer cells into the body of the rat, lipid vesicles were injected, and the growth rate of the cancer cells thereafter was investigated. First, it was found that injection of lipid vesicles to which ESA was not immobilized had the effect of suppressing the growth of cancer cells compared to the case where no injection was performed. Furthermore, it was confirmed that when the lipid vesicles on which ESA was immobilized were injected, the growth rate of the cancer cells was half that when the lipid vesicles on which ESA was not immobilized were injected.
次に、 本発明に係る遺伝子を固定化する方法の実施の形態を第 5図 を用いて説明する。 付着する遺伝子には、 プラスミ ド遺伝子として p c D A 3を骨格とし、 レポータージーンとしてルシフェラーゼ発現遗伝 子を組み込んだ p c D A 3 - 1 u c i を用いた。 このプラスミ ド DN Aを核酸結合性タンパクによりプラスリージェント （核酸結合性タンパ クと核移行シグナルの結合体） 処理を行った。 べシクルは構成脂質に 2 0〜 3 0重量％のカチオン性ペプチド脂質 （C P L) を混合することで、 カチオン性脂質含有脂質べシクルと して調製した。 ここで用いたカチォ ン性ペプチド脂質の構造は （CH3) 3 + (CHZ) 5 C ONHCH (C H 3) C ON [(C H2) 1 5 C H 3] 2 B r—であり、 N + C 5A 1 a 2 C i 6と略して表示される。 このべシクルをプラスリ一ジエンド処理 を行ったプラスミ ド DNAと混合し、 遺伝子をべシクルに固定化した。 このプラスミ ドー脂質べシクル複合体をターゲット細胞に接触させるこ とにより、 ターゲッ ト細胞において遺伝子を機能させ、 形質転換を行つ た。 以下、 実施例により本発明に係る遺伝子を固定化する方法について さらに詳細に説明する。 Next, an embodiment of the method for immobilizing a gene according to the present invention will be described with reference to FIG. As a gene to be attached, pcDA3-1uci having a backbone of pcDA3 as a plasmid gene and a luciferase expression gene incorporated as a reporter gene was used. The plasmid DNA was treated with a nucleic acid-binding protein at a plus reagent (combination of a nucleic acid-binding protein and a nuclear transport signal). Vesicles were prepared as cationic lipid-containing lipid vesicles by mixing 20 to 30% by weight of cationic peptide lipid (CPL) with the constituent lipids. Structure of Kachio emissions peptide lipid used here (CH 3) 3 + (CH Z) 5 C ONHCH (CH 3) C ON [(CH 2) 1 5 CH 3] is 2 B r-, N + is displayed abbreviated as C 5 a 1 a 2 C i 6. This vesicle was mixed with plasmid DNA that had been treated with plasmid end, and the gene was immobilized on the vesicle. By bringing this plasmid lipid vesicle complex into contact with a target cell, the gene was made to function in the target cell, and transformation was performed. Hereinafter, the method for immobilizing the gene according to the present invention will be described in more detail with reference to examples.
本実施例では、 脂質べシクルと して粒径が 1 μ ιη以上のいわゆるマ イク口サイズべシクルを用いた。 主成分と してソルビタンォレイン酸モ ノエステルを使用している。 ここで、 本発明に係る脂質べシクルには構 成脂質に対して 3 0重量％の割合の C P Lを調整してある。 生成された べシクル含有溶液を適宜生理食塩水で希釈しながら用いてプラスミ ド D N Aと混合し、 脂質べシクルに遺伝子を固定化させて、 これをターゲッ ト細胞である H e L a細胞に 3時間作用させた。 その後、 4 8時間おい た後、 基質物質を加え、 その発色を測定した。 遺伝子にはルシフェラ一 ゼ発現遺伝子を組み込んでいるため、 導入された遺伝子が機能し、 ルシ フェラ一ゼを発現すれば、 基質物質と反応して発色する。 In this example, a so-called lipid vesicle having a particle size of 1 μιη or more was used. A mouth-sized vesicle was used. Sorbitan oleic acid monoester is used as the main component. Here, the lipid vesicle according to the present invention is adjusted to have a CPL at a ratio of 30% by weight to the constituent lipid. The resulting vesicle-containing solution is mixed with plasmid DNA by appropriately diluting with physiological saline, and the gene is immobilized on lipid vesicles, which are then transferred to the target cells, HeLa cells. Time acted. After 48 hours, a substrate substance was added, and the color development was measured. Since the gene contains a luciferase-expressing gene, the introduced gene functions and, when luciferase is expressed, reacts with a substrate substance to develop color.
この結果を示すのが、 第 6図及び第 7図である。 縦軸はルシフェラ ーゼの活性量を表示し、 この値が高いほど導入された遺伝子が機能して、 ターゲッ ト細胞で形質転換が生じていることを示す。 図中で L ' A M I N Eと記されているのは比較例であり、 リボフェク トアミンプラス試薬 で処理されたプラスミ ドをリポソームに結合させたものの結果である。 第 6図中でスパン 8 0べシクルと示されるのは C P Lを混合していない 脂質べシクルの実験値である。 これによると、 C P Lなしべシクルでは. 遺伝子導入が十分に行われていない。 一方、 C P L入りべシクルでは、 遺伝子導入がよく行われ、 希釈の程度を適切にすることにより リポソ一 ムと同等以上の働きを有する。  Figures 6 and 7 show the results. The vertical axis indicates the amount of luciferase activity, and a higher value indicates that the introduced gene is functioning and that the target cell has been transformed. In the figure, L'AMINE is a comparative example, and is a result of binding of liposome to plasmid treated with ribofectamine plus reagent. In FIG. 6, what is indicated as span 80 vesicles is an experimental value of lipid vesicles not containing CPL. According to this, gene transfer was not sufficiently performed in vesicles without CPL. On the other hand, vesicles containing CPL are well transduced and have a function equivalent to or better than that of liposomes by appropriate dilution.
本発明に係る遺伝子固定化方法の第 2の実施例と して、 脂質べシク ルと して粒径が 1 O O n m以下のいわゆるナノサイズべシクルを用いた t その結果を第 8図に示す。 ナノサイズべシクルの場合でも遺伝子キヤリ ァと して機能していることがわかる。 ターゲッ ト細胞と して H e L a細 胞以外に M D A M B— 4 6 8 (ヒ ト乳がん細胞）、 C H O (チヤィニー ズハムスター卵巣細胞）、 C O S 7 (アフ リ カミ ドリザル腎臓由来細 胞） に対しても実験したが、 それぞれにおいて遺伝子キャリアと して機 差替 え 用 紙（規則 26) , „ As the second embodiment of genes immobilization method according to the present invention, the particle size as a lipid base consequent Le is shown in Figure 8 the t result with 1 OO nm or less called nano-sized base Shikuru . It can be seen that even a nano-sized vesicle functions as a gene carrier. Target cells include MDAMB-468 (human breast cancer cells), CHO (Chinese hamster ovary cells), and COS 7 (cells derived from African monkey kidney) in addition to HeLa cells. In each case, a replacement paper was used as a gene carrier (Rule 26). , „
1 6  1 6
能していることが確認された。 It was confirmed that it was working.
本発明に係る遺伝子固定化方法の第 3の実施例として、 核酸結合性 タンパクにヒス トン、 プロタミン又はポリ Lリジンを使用した例を示す c これらの核酸結合性タンパクは、 リボフエタ トァミンプラス試薬に比べ て安価で入手しやすい。 第 9図にヒス トンを用いたときの結果を示すが- 濃度を適切に調整して使用すれば、 高価なリボフェク トァミ ンプラス試 薬と比べても、 同等以上の効果を有することがわかる。 さらにヒス トン, プロタミン及びポリ Lリジンはそれぞれ単独で用いるよりも組み合わせ て使用すれば、 より効果的である。 組み合わせを変えて実験した結果を 第 1 0図に示す。 これより、 プロタミンとポリ Lリジンの組み合わせ、 およびヒス トン、 プロタミ ン、 ポリ L リ ジンの組み合わせが特に優れて いることがわかる。 これは、 C P L入りべシクルとしてマイクロサイズ を用いたときでも、 ナノマイクロサイズを用いたときでも同様である。 産業上の利用可能性 As a third embodiment of genes immobilization method according to the present invention, histone nucleic acid binding protein, protamine or poly-L-lysine c These nucleic acid binding protein that shows an example was used, as compared to Ribofueta Taminpurasu reagent Inexpensive and easy to obtain. Fig. 9 shows the results when the histone is used. It can be seen that the effect is equal to or higher than that of the expensive ribofectamine plus reagent when the concentration is appropriately adjusted and used. Furthermore, histone, protamine and poly-L-lysine are more effective when used in combination than when used alone. Figure 10 shows the results of experiments with different combinations. This indicates that the combination of protamine and poly-L-lysine and the combination of histone, protamine and poly-L-lysine are particularly excellent. This is the same whether micro-sized or nano-micro-sized vesicles containing CPL are used. Industrial applicability
本発明に係る脂質べシクル、 脂質べシクルの製造方法及び脂質べシ クルへ遺伝子を固定化する方法は、 癌等の病気の治療に有効な遺伝子や 薬剤を患部へ運ぶキヤリアを、 安価で安定な人工脂質で形成することが でき、 医薬品産業において利用価値の高いものである。 さらに、 体内の 食細胞に捕捉されにく く、 正常細胞を避けて癌細胞等の患部のみに作用 するので治療効果が高く、 しかも副作用が少ないという効果も有し、 癌 の遺伝子療法の利用性を広げるので、 医薬産業での適用範囲は広い。  The lipid vesicle, the method for producing the lipid vesicle, and the method for immobilizing a gene on the lipid vesicle according to the present invention are inexpensive and stable for carrying a gene or drug effective for treating a disease such as cancer to an affected area. It can be formed with various artificial lipids and has high utility value in the pharmaceutical industry. Furthermore, it is hard to be trapped by phagocytic cells in the body and acts only on the affected area, such as cancer cells, avoiding normal cells. The scope of application in the pharmaceutical industry is wide.

Claims

請 求 の 範 囲 The scope of the claims
1 . 一次乳化剤と してソルビタンエステルと溶媒を含む処理液に水を加 えて、 超音波照射を行って乳化させる工程と、 前記処理が行われた処理 液から溶媒を留去し、 これに二次乳化剤と してソルビタンエステルのェ チレンォキシド付加物を配合し、 乳化させる工程を含むことを特徴とす る脂質べシクルの製造方法。  1. A process in which water is added to a treatment solution containing a sorbitan ester and a solvent as a primary emulsifier, and the mixture is emulsified by ultrasonic irradiation, and the solvent is distilled off from the treatment solution subjected to the treatment. A method for producing a lipid vesicle, which comprises a step of mixing and emulsifying an ethylene oxide adduct of a sorbitan ester as a secondary emulsifier.
2 . コレステロールとレシチンを含む処理液の溶剤にへキサンを用い、 これに一次乳化剤と してソルビタンエステルを配合し、 水を加えて、 超 音波照射を行って乳化させる工程と、 前記処理が行われた処理液から溶 媒を留去し、 その残液に二次乳化剤と してソルビタンエステルのェチレ ンォキシド付加物を配合し、 攪拌させて乳化させる工程を含むことを特 徴とする脂質べシクルの製造方法。  2. Hexane is used as a solvent for the treatment liquid containing cholesterol and lecithin, and sorbitan ester is added as a primary emulsifier thereto, water is added, and the mixture is subjected to ultrasonic irradiation to emulsify the mixture. A lipid vesicle characterized by comprising a step of distilling off the solvent from the treated solution, adding a sorbitan ester ethylenoxide adduct as a secondary emulsifier to the remaining solution, and stirring and emulsifying the mixture. Manufacturing method.
3 . —次乳化剤と して、 ソルビタンォレイン酸モノエステルを用いるこ とを特徵とする請求項 1又は請求項 2に記載の脂質べシクルの製造方法 < 3. The method for producing a lipid vesicle according to claim 1 or 2, wherein sorbitan oleic acid monoester is used as the secondary emulsifier.
4 . 二次乳化剤と して、 ソルビタンォレイン酸モノエステルのエチレン ォキシド付加物を用いる請求項 1又は請求項 2に記載の脂質べシクルの 製造方法。 4. The method for producing a lipid vesicle according to claim 1 or 2, wherein an ethylene oxide adduct of sorbitan oleic acid monoester is used as the secondary emulsifier.
5 . 処理液を冷却しながら超音波照射を行って一次乳化させる請求項 1 乃至請求項 4に記載の脂質べシクルの製造方法。  5. The method for producing a lipid vesicle according to any one of claims 1 to 4, wherein the treatment liquid is cooled and subjected to ultrasonic irradiation to perform primary emulsification.
6 . メンブランろ過を行い、 粒径が 2 0 0 n m以下のナノサイズ脂質べ シクルを通過させる請求項 1乃至請求項 5に記載の脂質べシクルの製造 方法。 6. The method for producing a lipid vesicle according to any one of claims 1 to 5, wherein the filtration is performed by a membrane to pass a nano-sized lipid vesicle having a particle diameter of 200 nm or less.
7 . 請求項 1乃 請求項 6に記載の脂質べシクルの製造方法により製造 される脂質べシクル。  7. A lipid vesicle produced by the method for producing a lipid vesicle according to claim 6.
8 . プラスミ ド遺伝子に核酸結合性タンパクを加え、 カチオン性ぺプチ ド脂質を含む人工脂質より構成される脂質べシクルと混合することによ 1 o 8. Add a nucleic acid binding protein to the plasmid gene and mix it with a lipid vesicle composed of artificial lipids containing cationic peptide lipids. 1 o
り、 脂質べシクルに遺伝子を固定化する方法。 A method for immobilizing genes on lipid vesicles.
9 . 前記核酸結合性タンパクは、 ヒス トン、 プロタミン及びポリ Lリジ ンからなる群から選ばれることを特徴とする請求項 8に記載の遺伝子を 固定化する方法。  9. The method according to claim 8, wherein the nucleic acid binding protein is selected from the group consisting of histone, protamine, and poly-L lysine.
1 0 . 前記核酸結合性タンパクは、 ヒス トン、 プロタミン及びポリ Lリ ジンからなる群から選ばれる 2つ以上の核酸結合性タンパクの組み合わ せであることを特徴とする請求項 8に記載の遺伝子を固定化する方法。 10. The gene according to claim 8, wherein the nucleic acid binding protein is a combination of two or more nucleic acid binding proteins selected from the group consisting of histone, protamine and poly-L lysine. How to immobilize.
1 1 . 請求項 8乃至請求項 1 0に記載の遺伝子を固定化する方法により 遺伝子を固定化した脂質べシクル。 11. A lipid vesicle having a gene immobilized by the method for immobilizing a gene according to any one of claims 8 to 10.
1 2 . カチオン性ペプチド脂質を含む人工脂質と、 プラスミ ド遺伝子と. プラスミ ド遺伝子と、 ヒス トン、 プロタミン及びポリ Lリジンからなる 群から選ばれる核酸結合性タンパクを有する脂質べシクル。 12. A lipid vesicle having an artificial lipid containing a cationic peptide lipid, a plasmid gene, a plasmid gene, and a nucleic acid binding protein selected from the group consisting of histone, protamine and poly-L-lysine.
1 3 . —次乳化剤としてのソルビタンエステルを溶解した溶媒に水を加 えて処理液とし、 この処理液を冷却しながら超音波照射を行って乳化さ せる一次乳化工程と、 前記処理が行われた処理液から溶媒を留去し、 こ れに二次乳化剤としてソルビタンエステルのエチレンォキシド付加物と P E G脂質を配合して乳化させる二次乳化工程を含むことを特徴とする 脂質べシクルの製造方法。  1 3 .—A primary emulsification step in which water is added to a solvent in which a sorbitan ester as a secondary emulsifier is dissolved to form a treatment liquid, and the treatment liquid is cooled and subjected to ultrasonic irradiation to emulsify, and A method for producing a lipid vesicle, comprising a secondary emulsification step of evaporating a solvent by distilling off the solvent from the treatment solution and mixing the resulting mixture with a PEG lipid and an ethylene oxide adduct of a sorbitan ester as a secondary emulsifier. .
1 4 . 前記ソルビタンエステノレと してソノレビタンォレイン酸モノエステ ルを、 前記溶媒としてへキサンを用いる請求項 1 3に記載の脂質べシク ルの製造方法。  14. The method for producing a lipid vesicle according to claim 13, wherein sonorebitanoleic acid monoester is used as the sorbitan esterol, and hexane is used as the solvent.
1 5 . 前記 P E G脂質として脂質誘導体を持つ P E G脂質を用いる請求 項 1 3又は請求項 1 4に記載の脂質べシクルの製造方法。  15. The method for producing a lipid vesicle according to claim 13 or 14, wherein a PEG lipid having a lipid derivative is used as the PEG lipid.
1 6 . 一次乳化工程において処理液にさらにィソチオシアン酸ォクタデ シルエステル （ I A O E ) を混入するとともに、 二次乳化工程の後に、 可溶性プロティン Aを混入する工程と、 抗体を添加する工程を含むこと を特徴とする請求項 1 3乃至請求項 1 5に記載の脂質べシクルの製造方 法。 16. In the primary emulsification process, octadecyl isoisothiocyanate (IAOE) is further mixed into the treatment solution, and after the secondary emulsification process, a process of mixing soluble protein A and a process of adding an antibody are included. The method for producing a lipid vesicle according to any one of claims 13 to 15, wherein:
1 7. 請求項 1 3乃至請求項 1 5のいずれかに記載の脂質べシクルの製 造方法により製造される脂質べシクル。  1 7. A lipid vesicle produced by the method for producing a lipid vesicle according to any one of claims 13 to 15.
1 8. ソルビタンエステルで構成される膜と、 当該膜に付着した脂質誘 導体を持つ P E G脂質と、 プロテイン Aと結合した抗体と、 前記プロテ ィン Aを結合させる脂質アンカーのと してィソチオシアン酸ォクタデシ ルエステル （ I AO E) を有することを特徴とする脂質べシクル。  1 8. A membrane composed of sorbitan ester, a PEG lipid having a lipid derivative attached to the membrane, an antibody bound to protein A, and isothiothiocyanate as a lipid anchor for binding protein A A lipid vesicle comprising octadecyl ester (IAOE).
PCT/JP2002/006312 2001-06-22 2002-06-24 Lipid vesicles, process for producing lipid vesicles and method of immobilizing gene on lipid vesicles WO2003000291A1 (en)

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JP2001190518A JP2003001097A (en) 2001-06-22 2001-06-22 Method of making nanosize lipid vesicle
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