WO2003000291A1 - Vesicules lipidiques, processus de production de vesicules lipidiques et procede d'immobilisation de gene sur ces vesicules lipidiques - Google Patents

Vesicules lipidiques, processus de production de vesicules lipidiques et procede d'immobilisation de gene sur ces vesicules lipidiques 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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English (en)
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/ja
Priority claimed from JP2001190518A external-priority patent/JP2003001097A/ja
Priority claimed from JP2001266659A external-priority patent/JP4928689B2/ja
Application filed by Techno Network Shikoku Co., Ltd. filed Critical Techno Network Shikoku Co., Ltd.
Publication of WO2003000291A1 publication Critical patent/WO2003000291A1/fr

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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

La présente invention concerne des vésicules lipidiques de petite taille qu'on peut obtenir par un processus d'émulsification en deux étapes comprenant l'étape de l'addition d'eau à une liqueur de traitement contenant un ester sorbitane en guise d'émulsifiant principal et un solvant et la formation d'une émulsion par sonication aux ultrasons et, l'étape de distillation du solvant hors de cette liqueur ainsi prétraitée et de l'addition d'un oxyde éthylène d'ester sorbitane en guise d'émulsifiant secondaire suivie de l'émulsification. Le lipide artificiel stable le moins onéreux permet d'obtenir un porteur non viral par lequel on peut apporter efficacement un médicament sur une partie affectée sans que celui-ci ne soit capturé par des phagocytes in vivo. Par l'utilisation d'un lipide peptidique cationique et l'addition d'un gène plasmide et d'une protéine de liaison à l'acide nucléique sur celui-ci, le gène peut être efficacement immobilisé sur la vésicule lipidique et appliqué à une thérapie génique.
PCT/JP2002/006312 2001-06-22 2002-06-24 Vesicules lipidiques, processus de production de vesicules lipidiques et procede d'immobilisation de gene sur ces vesicules lipidiques WO2003000291A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
JP2001-190515 2001-06-22
JP2001190515A JP2003012503A (ja) 2001-06-22 2001-06-22 脂質ベシクルおよび遺伝子を固定化する方法
JP2001190518A JP2003001097A (ja) 2001-06-22 2001-06-22 ナノサイズ脂質ベシクルの製造方法
JP2001-190518 2001-06-22
JP2001266659A JP4928689B2 (ja) 2001-09-04 2001-09-04 脂質ベシクルおよび脂質ベシクルの製造方法
JP2001-266659 2001-09-04

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Citations (6)

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Publication number Priority date Publication date Assignee Title
JPS6293223A (ja) * 1985-10-18 1987-04-28 Meiji Milk Prod Co Ltd 注射用ｗ／ｏ／ｗ型複合エマルジヨン及びその製造法
WO1991000289A2 (fr) * 1989-06-23 1991-01-10 The Liposome Company, Inc. Liposomes cibles et procedes d'accouplement de liposomes-proteines
JPH06329558A (ja) * 1993-05-25 1994-11-29 Eisai Co Ltd 合成脂質リポソーム
WO1999033493A1 (fr) * 1997-12-23 1999-07-08 Inex Pharmaceuticals Corporation Oligomeres polyamidiques
WO2000015825A1 (fr) * 1997-03-28 2000-03-23 Alza Corporation Complexe de plasmide-liposome condense pour transfection
EP1097721A2 (fr) * 1999-11-05 2001-05-09 Nof Corporation Adjuvant pour vaccin à base d'huile

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6293223A (ja) * 1985-10-18 1987-04-28 Meiji Milk Prod Co Ltd 注射用ｗ／ｏ／ｗ型複合エマルジヨン及びその製造法
WO1991000289A2 (fr) * 1989-06-23 1991-01-10 The Liposome Company, Inc. Liposomes cibles et procedes d'accouplement de liposomes-proteines
JPH06329558A (ja) * 1993-05-25 1994-11-29 Eisai Co Ltd 合成脂質リポソーム
WO2000015825A1 (fr) * 1997-03-28 2000-03-23 Alza Corporation Complexe de plasmide-liposome condense pour transfection
WO1999033493A1 (fr) * 1997-12-23 1999-07-08 Inex Pharmaceuticals Corporation Oligomeres polyamidiques
EP1097721A2 (fr) * 1999-11-05 2001-05-09 Nof Corporation Adjuvant pour vaccin à base d'huile

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Title
HIROSHI TERADA, TETSURO YOSHIMURA: "Lefe science no okeru liposome - jikken manual", 1 August 1992, SPRINGER-VERLAG TOKYO KABUSHIKI KAISHA, pages: 81, XP002960426 *
MASAYUKI NAKAGAKI: "Gendai butsuri kagaku koza 9, hyomen to colloid jotai", 10 July 1968, TOKYO KAGAKU DOJIN, pages: 243 - 244 *
TAMOTSU KONDO: "Gendai colloid kagaku", SANKYO SHUPPAN KABUSHIKI KAISHA, 30 May 1980 (1980-05-30), pages 64 - 68, XP002960427 *

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