WO2007091661A1 - Liposome modifié par une chaîne de sucre convenant pour l'imagerie moléculaire et utilisation et production de celui-ci - Google Patents

Liposome modifié par une chaîne de sucre convenant pour l'imagerie moléculaire et utilisation et production de celui-ci Download PDF

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WO2007091661A1
WO2007091661A1 PCT/JP2007/052289 JP2007052289W WO2007091661A1 WO 2007091661 A1 WO2007091661 A1 WO 2007091661A1 JP 2007052289 W JP2007052289 W JP 2007052289W WO 2007091661 A1 WO2007091661 A1 WO 2007091661A1
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Prior art keywords
ribosome
solution
sugar chain
buffer
group
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PCT/JP2007/052289
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English (en)
Japanese (ja)
Inventor
Noboru Yamazaki
Kazunori Oie
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National Institute Of Advanced Industrial Science And Technology
Katayama Chemical Industries Co., Ltd.
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Priority to JP2007557899A priority Critical patent/JPWO2007091661A1/ja
Publication of WO2007091661A1 publication Critical patent/WO2007091661A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0076—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion
    • A61K49/0084—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion liposome, i.e. bilayered vesicular structure
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a ribosome.
  • the ribosome of the present invention is a drug delivery system for recognizing target cells' tissues such as cancer and locally delivering drugs and genes to affected areas, which can be applied in bi-technology, particularly molecular imaging. It can be used as a cell / tissue sensing probe for diagnosis.
  • DDS drug and gene delivery system
  • the nanotechnology / materials promotion strategy of the Council for Science and Technology in Japan also includes “Nanobiology that uses and controls the mechanisms of materials and organisms” as an important area, and is one of the five years of research and development goals.
  • NNI National Nanotechnology Strategy
  • DDS drug and gene delivery system
  • the nanotechnology / materials promotion strategy of the Council for Science and Technology in Japan also includes “Nanobiology that uses and controls the mechanisms of materials and organisms” as an important area, and is one of the five years of research and development goals.
  • One example is “Establishment of basic seeds for biofunctional materials and pinpoint treatment technologies for extending health and life expectancy”.
  • the incidence and mortality of cancer has been increasing year by year as it becomes an aging society, and the development of target-oriented DDS, which is a novel therapeutic material, is awaited.
  • the importance of targeted DDS nanomaterials with no side effects in other diseases is drawing attention, and the scale of the field is expected to exceed 10 trillion yen in the near future.
  • glycolipids of glycolipids and glycoproteins are involved in various cell-to-cell communications such as the generation and morphogenesis of biological tissues, cell proliferation and differentiation, biological defense and fertilization mechanisms, canceration and its metastasis mechanism. It is becoming clear that it plays an important role as an information molecule.
  • peptidic drugs are generally water-soluble and have a high molecular weight, and the gastrointestinal tract has a low permeability to the small intestinal mucosa. Therefore, research on ligand-bound ribosomes is attracting attention as a DDS material for delivering these high molecular weight pharmaceuticals and genes into the blood of the intestinal tract (see Non-Patent Document 8).
  • Patent Document 1 discloses a pharmaceutical composition having a pharmaceutically acceptable carrier and a compound containing a component that selectively binds to a selectin receptor.
  • a sugar chain intended for oral administration is used as a pharmaceutical agent itself for inhibiting inflammatory diseases and other diseases mediated by cell adhesion. Is different.
  • the present inventors have developed a sugar chain-modified ribosome in which a sugar chain is bound to a ribosome via a linker protein (Patent Document 2). Furthermore, it was found that the type of sugar chain and the amount of sugar chain binding seem to be related to directivity to each target cell or target tissue (Patent Documents 3 to 4 and Non-Patent Documents 9 and 10). However, to date, no sugar chain-modified ribosome that is optimal for molecular imaging has been developed. In addition, there has been no systematic study on sugar chains useful in molecular imaging, and it has remained unclear what kind of sugar chains should be used.
  • Patent Literature 1 Japanese Patent Publication No. 5-507519
  • Patent Document 2 Japanese Patent Laid-Open No. 2003-226638
  • Patent Document 3 Japanese Patent Laid-Open No. 2003-226647
  • Patent Document 4 Pamphlet of International Publication No. 2005Z011632
  • Patent Document 5 Pamphlet of International Publication No. 2005Z011633
  • Non-patent literature l Yamazaki, N., Kojima, S., Bovin, ⁇ . V., Andre, S., Gabius, S. and Gabius, H. —J. (2000) Adv. Drug Delivery Rev. 43 , 225— 24
  • Non-Patent Document 2 Yamazaki, N., Jigami, ⁇ ., Gabius, ⁇ . —J., Kojima, S (200 1) Trends in Glycoscience and Glycotechnology 13, 319— 329. http: / / www. Gak. Co .jp / TIGG / 71PDF / yamazaki.pdf
  • Non-Patent Document 3 Forssen, E. and Willis, M. (1998) Adv. Drug Delivery Rev. 29, 249-271.
  • Non-Patent Document 4 DeFrees, S. A., Phillips, L., Guo, L. and Zalipsky, S. (19 96) J. Am. Chem. Soc. 118, 6101— 6104.
  • Non-Patent Document 5 Spevak, W., Foxall, C., Charych, D.H., Dasqupta, F. and
  • Non-Patent Document 6 Stahn, R., Schafer, H., Kernchen, F. and Schreiber, J. (1 998) Glycobiology 8, 311—319.
  • Non-Patent Document 7 Yamazaki, N., Jigami, Y., Gabius, H. —J., Kojima, S (200 1) Trends in Glycoscience and Glycotechnology 13, 319— 329. http: / / www. Gak. Co .jp / TIGG / 71PDF / yamazaki.pdf
  • Non-Patent Document 8 Lehr, C. — M. (2000) J. Controlled Release 65, 19—29
  • Non-Patent Document 9 Noboru Yamayose (2005), Development of Active 'Targeting DDS Nanoparticles, Journal of Nano Society, 3, 97- 102
  • Non-Patent Document 10 Noboru Yamayori (2006), Huarmasia, No. 42, No. 2, 2-6
  • an object of the present invention is to provide a sugar chain-modified ribosome useful for molecular imaging, and a drug delivery medium in which a drug or gene is encapsulated in the sugar chain-modified ribosome.
  • the present inventors have conducted intensive research and found that ribosomes whose ribosome surface has been modified with specific sugar chains are useful preparations for molecular imaging.
  • the present invention has been completed.
  • the present invention also provides a method for producing a sugar chain-modified ribosome useful for molecular imaging and a method for using the same.
  • the present invention provides, for example, the following means.
  • a sugar chain-modified ribosome the sugar chain-modified ribosome
  • the linker protein group binds to the outer surface of the ribosome
  • the sugar chain group binds to at least part of the linker protein group
  • the linker protein group binds to the outer surface of the ribosome or part of the linker protein group.
  • the sugar chain-modified ribosome includes structure I and structure ⁇ ,
  • X is capable of CH—NH binding with the linker protein contained in the ribosome.
  • R 1 is the linker protein group
  • R 2 is a linker-protein cross-linking group
  • R 3 is the sugar chain group
  • Y is a group in which the functional group b is removed from the structural unit force including the hydrophilic compound cross-linking group contained in the ribosome and the functional group b capable of peptide bonding;
  • R 4 is the hydrophilic compound crosslinking group
  • R 5 is the hydrophilic I ⁇ product groups, glycosylation ribosome of claim 1.
  • (Item 3) Item 2.
  • Item 4 The sugar chain-modified ribosome according to Item 3, wherein the fluorescence is imparted by a fluorescent dye compatible with the ribosome.
  • the fluorescent dye is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoe
  • the sugar chain-modified liposome according to Item 4 which is selected from the group consisting of 0, Alexa Fluor750 and fluorescein—4-isothiocyanate (FITC) and their combination.
  • the fluorescent dye is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoe
  • R 1 is a mammalian derived protein groups, glycosylation ribosome of claim 2. (Item 9)
  • R 1 is a human-derived protein groups, glycosylation ribosome of claim 8.
  • R 1 is a human-derived serum protein groups, glycosylation ribosome of claim 9. (Item 11)
  • R 1 is a sugar chain modification ribosome of claim 8, which is a serum albumin group.
  • R 2 is a 3, 3, 1 dithiobis (sulfosuccinimidyl propionate) group, bissulfo Succinimidyl suberate group, disuccinimidyl glutarate group, dithiobis succinimidyl propionate group, disuccinimidyl suberate group, ethylene glycol bis succinimidyl succinate group and ethylene Item 3.
  • the sugar chain-modified ribosome according to item 2 which is selected from the group consisting of glycol bissulfosuccinimidyl succinate group.
  • Item 13 The sugar chain-modified ribosome according to Item 12, wherein R 2 is a 3, 3, -dithiobis (sulfosuccinimidyl propionate) group.
  • R 3 is Shiariruruisu X group, N- ⁇ cetyl lactosamine group, alpha 1-6 mannobiose group is selected from the group consisting a combination force thereof, glycosylation liposome of claim 2.
  • R 3 is Shiariruruisu X groups include a modified bond density of said Shiariruruisu X groups force 0. OOOlmg sugar Zm g lipid ⁇ 500 mg sugar Zmg range of lipids, glycosylation ribosome of claim 14.
  • R 3 is an N-acetyllactosamine group, and the N-acetyllactosamine group has a modified bond density ranging from 0.0 OOlmg sugar chain Zmg lipid to 500 mg sugar chain Zmg lipid.
  • Item 14 is that wherein R 3 is an ⁇ 1-6 mannobiose group, and the ⁇ 1-6 mannobiose group has a modified bond density in the range of 0.0001 mg sugar chain Zmg lipid to 500 mg sugar chain Zmg lipid.
  • R 4 is a bis (sulfosuccinimidyl) suberate group, a disuccinimidyl glutarate group, a dithiobissuccinimidyl propionate group, a disuccinimidyl suberate group, 3 , 3, 1 dithiobis (sulfosuccinimidyl propionate) group, ethylene glycol bissuccinimidyl succinate group and ethylene glycol bissulfosuccinimid Item 3.
  • the sugar chain-modified ribosome according to Item 2 which is selected from a rusuccinate group.
  • R 4 is bis (sulfosuccinimidyl I succinimidyl) scan base rate group, glycosylation ribosome of claim 18.
  • Item 3 The sugar chain-modified ribosome according to Item 2, wherein R 5 is a tris (hydroxyalkyl) alkylamino group.
  • Tris (hydroxyalkyl) alkylamino group Hydroxyalkyl is C to C hydride
  • Item 24 The sugar chain-modified ribosome according to Item 23, wherein X is redesignoside.
  • Item 3 The sugar chain-modified ribosome according to Item 2, wherein the functional group b is an amino group.
  • Item 26 The sugar chain-modified liposome according to Item 25, wherein Y is phosphatidylethanolamine.
  • the sugar chain-modified ribosome according to item 1, comprising the ribosome force dipalmitoyl phosphatidylcholine, cholesterol, ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine and sodium cholate.
  • the item 27 comprising the ribosomal force dipalmitoyl phosphatidylcholine, cholesterol, ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine and sodium cholate in a molar ratio of 35: 40: 15: 5: 5: 167.
  • Sugar chain-modified ribosomes
  • R 2 is a 3, 3, 1 dithiobis (sulfosuccinimidyl propionate) group
  • R 3 is Shiariruruisu X group, N- ⁇ cetyl lactosamine group, alpha 1-6 mannobiose group is selected from the group consisting a combination force thereof, glycosylation liposome of claim 2.
  • R 3 is selected from the group consisting of a sialyl Lewis X group, a ⁇ -acetyl lactosamine group, an ⁇ 1-6 mannobiose group, and a combination force thereof, and
  • Item 3 The sugar chain-modified ribosome according to Item 2, wherein R 4 is a bis (sulfosuccinimidyl) suberate group.
  • R 2 is a 3,3,1 dithiobis (sulfosuccinimidylpropionate) group
  • R 3 is a sialyl Lewis X group, a ⁇ -acetyllactosamine group, an ⁇ 1-6 mannobiose group, and Selected from the group of their combination power
  • Item 3 The sugar chain-modified ribosome according to Item 2, wherein R 4 is a bis (sulfosuccinimidyl) suberate group.
  • the ribosomal strength includes dipalmitoyl phosphatidylcholine, cholesterol, ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine and sodium cholate,
  • R 2 is a 3,3,1-dithiobis (sulfosuccinimidylpropionate) group
  • R 3 is a sialyl Lewis X group, a ⁇ -acetyllactosamine group, ⁇ 1-6 mannobiose Selected from the group consisting of groups and their combination powers, and
  • Item 3 The sugar chain-modified ribosome according to Item 2, wherein R 4 is a bis (sulfosuccinimidyl) suberate group.
  • the ribosomal strength includes dipalmitoyl phosphatidylcholine, cholesterol, ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine and sodium cholate,
  • R 1 is a serum albumin group
  • R 2 is a 3,3,1 dithiobis (sulfosuccinimidylpropionate) group
  • R 3 is a sialyl Lewis X group, an N-acetyllactosamine group, an ⁇ 1-6 mannobiose group, and Selected from the group consisting of those combinations
  • R 4 is a bis (sulfosuccinimidyl) suberate group
  • R 5 is a tris (hydroxymethyl) Aminometan group, glycosylation liposome of claim 2.
  • the sugar chain-modified ribosome is
  • the ratio of protein to lipid is about 0.1 to about 0.5.
  • Item 38 An imaging agent comprising the sugar chain-modified ribosome according to any one of items 1 to 37. (Item 39)
  • composition for delivering a substance to a desired site comprising the sugar chain-modified ribosome according to any one of items 1 to 37 and a substance desired to be delivered.
  • composition of item 39, wherein the desired substance is a diagnostic agent or a research reagent.
  • composition according to item 40 wherein the diagnostic agent is selected from the group consisting of a DNA probe diagnostic agent, a tumor diagnostic agent, a hematological test reagent, and a microbiological test reagent.
  • composition of item 39 for use in molecular or in vivo imaging.
  • composition of item 39, wherein the substance comprises the biological agent comprises the biological agent.
  • a pharmaceutical composition further comprising the sugar chain-modified ribosome according to any one of items 1 to 37 and a pharmaceutically active ingredient.
  • Item 4 wherein the pharmaceutically active ingredient is an agent for treating a disease in the brain, liver, kidney, spleen, lung, spleen or heart, or an agent for treating inflammation or tumor. 4.
  • the desired site is selected from the group consisting of brain, liver, kidney, spleen, lung, spleen, heart, inflammatory site and tumor site force.
  • a method for labeling a desired site comprising:
  • the method includes the step of administering to the subject a composition for labeling the desired site, wherein the composition comprises the sugar chain-modified ribosome according to any one of items 1 to 37 and a pharmaceutically acceptable product.
  • the desired site is selected from the group consisting of brain, liver, kidney, spleen, lung, spleen, heart, inflammatory site and tumor site force.
  • a method for producing a sugar chain-modified ribosome comprising:
  • the fluorescent labeling solution of step (c) is [Chem. 10-5] Ku5. 5>
  • Item 52 The method according to Item 49, wherein the linker protein power in step (e) is human serum albumin.
  • a method for producing a sugar chain-modified ribosome for delivering a drug to a target delivery site comprising:
  • a method according to item 53 comprising the step of filter filtration subsequent to the step D).
  • the fluorescent dye solution in the step (A4) is a fluorescent dye standard labeled with a fluorescent dye.
  • Fluorescent labels can bind proteins ZN- tris (hydroxymethyl) 3 Aminobu port pan sulfonic acid buffer (P H8. 4) Fluorescent dyes ZN tris (hydroxymethyl) To a solution of 3- ⁇ amino propane sulfonic acid buffer mixing the (pH 8.4) solution and stirring at room temperature to about 37 ° C; and
  • step (2) A step of ultrafiltration of the mixed solution in step (1) with a molecular weight cut-off of 10,000 to remove the free fluorescent dye
  • Step B) includes the following:
  • (B2) Add bis (sulfosuccinimidyl) suberate to the solution in which the buffer solution has been converted to the carbonate buffer solution in the step (B1), and refrigerate to about 37 ° C. Agitation and ultrafiltration at a molecular weight cut off of 300,000 to remove the free bis (sulfosuccinimidyl) suberate;
  • step (B3) a solution obtained by removing the free bis (sulfosuccinimidyl) suberate is added to 330 mM Tris (hydroxymethyl) aminomethane Z carbonate buffer ( ⁇ 8. 5) Add the solution, stir at refrigeration to about 37 ° C, stir at refrigeration to room temperature overnight, and ultrafilter with a molecular weight cut off of 300,000 to remove free tris (hydroxymethyl) aminomethane. And replacing the carbonate buffer with N-tris (hydroxymethyl) -3-aminopropanesulfonate buffer (pH 8.4) to produce a solution containing ribosomes that have been hydrophilically treated;
  • Step B) includes the following:
  • step (B3,) In the step (B2,), in the solution from which the free bis (sulfosuccinimidyl) suberate was removed, 330 mM tris (hydroxymethyl) aminomethane / carbonate buffer (pH 8.5) was added. ) Add the solution, stir at refrigeration to approximately 37 ° C, stir at refrigeration to room temperature overnight, and centrifuge twice at a molecular weight cut off of 100,000, 2000 x g for 60 minutes. Ultrafiltration is performed to remove free tris (hydroxymethyl) aminomethane, and the carbonate buffer is replaced with N-tris (hydroxymethyl) -3-amaminopropanesulfonate buffer (pH 8.4). Producing a solution containing ribosomes that have been treated with hydrophilicity and hydrophilicity;
  • Step C) includes the following:
  • step (C2) the solution containing the ribosome whose surface is oxidized is ultrafiltered with a molecular weight cut off of 300,000 to remove the free sodium metaperiodate, and the N— Replacing tris (hydroxymethyl) -3-aminopropanesulfonate buffer with PBS buffer (pH 8.0);
  • step (C3) human serum albumin / PBS buffer (pH 8.0) is added to the solution in which the buffer is exchanged with the PBS buffer, and the reaction is performed at refrigeration to room temperature.
  • Step C) includes the following:
  • step (C2) the solution containing the ribosome whose surface is oxidized is ultrafiltered with a molecular weight cut off of 300,000 to remove the free sodium metaperiodate, and the N— Replacing tris (hydroxymethyl) -3-aminopropanesulfonate buffer with PBS buffer (pH 8.0);
  • step (C3) human serum albumin / PBS buffer (pH 8.0) is added to the solution in which the buffer is exchanged with the PBS buffer, and the reaction is performed at refrigeration to room temperature.
  • reaction solution is stirred at refrigeration to room temperature, ultrafiltered with a molecular weight cut off of 300,000 to remove free human serum albumin, and the buffer solution of the solution is changed to a carbonate buffer solution (pH 8). .5) Replacement process
  • Step C) includes the following:
  • the solution containing the ribosome with an oxidized surface of the particle is separated twice with a molecular weight cutoff of 100,000, 2000 ⁇ g for 60 minutes. Removing the free sodium metaperiodate and replacing the N-tris (hydroxymethyl) 3-aminopropanesulfonate buffer solution with PBS buffer (pH 8.0). ;
  • step (C3 ′) human serum albumin / PBS buffer (pH 8.0) was added to the solution in which the buffer was replaced with the PBS buffer, and the reaction was performed at refrigeration to room temperature. Producing a solution;
  • Step C) includes the following:
  • step (C3 ′) human serum albumin / PBS buffer (pH 8.0) was added to the solution in which the buffer was replaced with the PBS buffer, and the reaction was performed at refrigeration to room temperature. Producing a solution;
  • reaction solution was stirred at refrigeration to room temperature, ultrafiltered by centrifugation twice under conditions of a molecular weight cut off of 100,000 and 200 OX g for 60 minutes, and the human serum A 60.
  • Step D) includes the following:
  • (D1) a step of dissolving the sugar chain in purified water and reacting at room temperature to about 37 ° C. under saturated ammonium bicarbonate to prepare an amino sugar chain solution;
  • step (D3) the aminated sugar chain solution is added to the solution from which the free 3,3,4-dithiobis (sulfosuccinimidyl propionate) has been removed, and the mixture is refrigerated to about 37 ° C. React with C, add tris (hydroxymethyl) aminomethane Z carbonate buffer (PH8.5), stir overnight at refrigerated ⁇ 37 ° C, ultrafilter with molecular weight cut off 300,000, Removing the sugar chain and the tris (hydroxymethyl) aminomethane;
  • step (D4) a step of replacing the buffer solution of the solution from which the free sugar chain and tris (hydroxymethyl) aminomethane have been removed in the step (D3) with a HEPES buffer solution (pH 7.2),
  • a method for producing a fluorescent dye-containing sugar chain-modified ribosome comprising:
  • Step A) includes the following:
  • the fluorescent dye solution containing the fluorescent dye is mixed with the solution sonicated in step (A3), and the mixed solution is ultrafiltered with a molecular weight cut off of 10,000 to enclose the fluorescent dye.
  • the fluorescent dye solution is added to human serum albumin ZN—Tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) solution with fluorescent dye / N-Tris ( Hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) solution was mixed, stirred at 37 ° C, and ultrafiltered with a molecular weight cut off of 10,000 to remove the free fluorescent dye.
  • Step B) includes the following:
  • step (B2) Add bis (sulfosuccinimidyl) suberate to the solution in which the buffer solution has been converted to the carbonate buffer solution in the step (B1), and refrigerate to about 37 ° C. And ultrafiltration with a molecular weight cut off of 300,000 to remove the free bis (sulfosuccinimidyl) suberate; and (B3) In the step (B2), a solution obtained by removing the free bis (sulfosuccinimidyl) suberate is added to 330 mM Tris (hydroxymethyl) aminomethane Z carbonate buffer ( ⁇ 8.
  • Step C) includes the following:
  • step (C2) the solution containing the ribosome whose surface is oxidized is ultrafiltered with a molecular weight cut off of 300,000 to remove the free sodium metaperiodate, and the N— Replacing tris (hydroxymethyl) -3-aminopropanesulfonate buffer with PBS buffer (pH 8.0);
  • step (C3) human serum albumin / PBS buffer (pH 8.0) is added to the solution in which the buffer is exchanged with the PBS buffer, and the reaction is performed at refrigeration to room temperature. Producing steps; and
  • Step D) includes the following:
  • (D1) a step of dissolving the sugar chain in purified water and reacting at room temperature to about 37 ° C under ammonium hydrogen carbonate saturation to prepare an amino sugar chain solution;
  • step (D3) the aminated sugar chain solution is added to the solution from which the free 3,3,4-dithiobis (sulfosuccinimidyl propionate) has been removed, and the mixture is refrigerated to about 37 ° C. React with C, add tris (hydroxymethyl) aminomethane Z carbonate buffer (PH8.5), stir overnight at refrigerated ⁇ 37 ° C, ultrafilter with molecular weight cut off 300,000, Removing sugar chains and the tris (hydroxymethyl) aminomethane; and
  • step (D4) In the step (D3), including the step of replacing the buffer solution of the solution from which the free sugar chain and the tris (hydroxymethyl) aminomethane have been removed with a HEPES buffer solution (pH 7.2), The manufacturing method as described.
  • Step A) includes the following:
  • step (A4) The fluorescent dye solution containing the fluorescent dye is mixed with the solution sonicated in step (A3), and the mixed solution is ultrafiltered with a molecular weight cut off of 10,000 to enclose the fluorescent dye.
  • the fluorescent dye solution is added to human serum albumin ZN—Tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) solution with fluorescent dye / N-Tris ( Hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) solution was mixed, stirred at 37 ° C, and ultrafiltered with a molecular weight cut off of 10,000 to remove the free fluorescent dye.
  • a step prepared by the step of Step B) includes the following:
  • step (B3) a solution obtained by removing the free bis (sulfosuccinimidyl) suberate is added to 330 mM Tris (hydroxymethyl) aminomethane Z carbonate buffer ( ⁇ 8. 5) Add the solution, stir at refrigeration to about 37 ° C, stir at refrigeration to room temperature overnight, ultrafilter with a fractional fraction of 300,000, and free tris (hydroxymethyl) aminomethane. And removing the carbonate buffer with N-tris (hydroxymethyl) 3-aminopropanesulfonate buffer (PH8.4) to produce a solution containing ribosomes that have been treated with hydrophilic acid. And
  • Step C) includes the following:
  • step (C2) the solution containing the ribosome whose surface is oxidized is ultrafiltered with a molecular weight cut off of 300,000 to remove the free sodium metaperiodate, and the N— Replacing tris (hydroxymethyl) -3-aminopropanesulfonate buffer with PBS buffer (pH 8.0);
  • step (C3) human serum albumin / PBS buffer (pH 8.0) is added to the solution in which the buffer is exchanged with the PBS buffer, and the reaction is performed at refrigeration to room temperature. Producing steps; and
  • reaction solution was stirred at refrigeration to room temperature, and the molecular weight cutoff was 300,000. Filtering, removing the free human serum albumin and replacing the buffer of the solution with carbonate buffer (PH8.5),
  • Step D) includes the following:
  • (D1) a step of dissolving the sugar chain in purified water and reacting at room temperature to about 37 ° C under ammonium hydrogen carbonate saturation to prepare an amino sugar chain solution;
  • step (D3) the aminated sugar chain solution is added to the solution from which the free 3,3,4-dithiobis (sulfosuccinimidyl propionate) has been removed, and the mixture is refrigerated to about 37 ° C. React with C, add tris (hydroxymethyl) aminomethane Z carbonate buffer (PH8.5), stir overnight at refrigerated ⁇ 37 ° C, ultrafilter with molecular weight cut off 300,000, Removing sugar chains and the tris (hydroxymethyl) aminomethane; and
  • step (D4) In the step (D3), including the step of replacing the buffer solution of the solution from which the free sugar chain and the tris (hydroxymethyl) aminomethane have been removed with a HEPES buffer solution (pH 7.2), The manufacturing method as described.
  • Step A) includes the following:
  • step (A3) stirring the resuspension at 30-40 ° C, purging with nitrogen, and sonicating; and (A4)
  • the fluorescent dye solution containing the fluorescent dye is mixed with the solution sonicated in step (A3), and the mixed solution is ultrafiltered with a molecular weight cut off of 10,000 to enclose the fluorescent dye.
  • the fluorescent dye solution is added to human serum albumin ZN—Tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) solution with fluorescent dye / N-Tris ( Hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) solution was mixed, stirred at 37 ° C, and ultrafiltered with a molecular weight cut off of 10,000 to remove the free fluorescent dye.
  • Step B) includes the following:
  • step (B3,) In the step (B2,), in the solution from which the free bis (sulfosuccinimidyl) suberate was removed, 330 mM tris (hydroxymethyl) aminomethane / carbonate buffer (pH 8.5) was added. ) Add the solution, stir at refrigeration to approximately 37 ° C, stir at refrigeration to room temperature overnight, and centrifuge twice at a molecular weight cut off of 100,000, 2000 x g for 60 minutes. Ultrafiltration is performed to remove free tris (hydroxymethyl) aminomethane, and the carbonate buffer is replaced with N-tris (hydroxymethyl) -3-amaminopropanesulfonate buffer (pH 8.4). And a step of producing a solution containing ribosome treated with hydrophilicity, wherein the step C) includes the following:
  • (D1) a step of dissolving the sugar chain in purified water and reacting at room temperature to about 37 ° C under ammonium hydrogen carbonate saturation to prepare an amino sugar chain solution;
  • step (D2) the aminated sugar chain solution is added to the solution from which the free 3, 3, 1 dithiobis (sulfosuccinimidyl propionate) has been removed, and the mixture is refrigerated to about 37 ° C. React with C, add tris (hydroxymethyl) aminomethane Z carbonate buffer (PH8.5), stir overnight at refrigerated ⁇ 37 ° C, ultrafilter with molecular weight cut off 300,000, Removing sugar chains and the tris (hydroxymethyl) aminomethane; and
  • step (D4) In the step (D3), including the step of replacing the buffer solution of the solution from which the free sugar chain and the tris (hydroxymethyl) aminomethane have been removed with a HEPES buffer solution (pH 7.2), The manufacturing method as described.
  • Step A) includes the following:
  • the fluorescent dye solution containing the fluorescent dye is mixed with the solution sonicated in step (A3), and the mixed solution is ultrafiltered with a molecular weight cut off of 10,000 to enclose the fluorescent dye.
  • the fluorescent dye solution is added to human serum albumin ZN—Tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) solution with fluorescent dye / N-Tris ( Hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) solution was mixed, stirred at 37 ° C, and ultrafiltered with a molecular weight cut off of 10,000 to remove the free fluorescent dye.
  • Step B) includes the following:
  • step (B3,) In the step (B2,), in the solution from which the free bis (sulfosuccinimidyl) suberate was removed, 330 mM tris (hydroxymethyl) aminomethane / carbonate buffer (pH 8.5) was added. ) Add the solution, stir at refrigeration to approximately 37 ° C, stir at refrigeration to room temperature overnight, and centrifuge twice at a molecular weight cut off of 100,000, 2000 x g for 60 minutes.
  • step (C3 ′) human serum albumin / PBS buffer (pH 8.0) was added to the solution in which the buffer was replaced with the PBS buffer, and the reaction was performed at refrigeration to room temperature. Producing a solution;
  • reaction solution was stirred at refrigeration to room temperature, ultrafiltered by centrifugation twice under conditions of a molecular weight cut off of 100,000 and 200 OX g for 60 minutes, and the human serum Removing the albumin and replacing the buffer of the solution with carbonate buffer (pH 8.5);
  • Step D) includes the following:
  • (D1) a step of dissolving the sugar chain in purified water and reacting at room temperature to about 37 ° C under ammonium hydrogen carbonate saturation to prepare an amino sugar chain solution;
  • step (D2) the aminated sugar chain solution is added to the solution from which the free 3, 3, 1 dithiobis (sulfosuccinimidyl propionate) has been removed, and the mixture is refrigerated to about 37 ° C. React with C, add tris (hydroxymethyl) aminomethane Z carbonate buffer (PH8.5), Refrigerated to 37 ° C overnight, ultrafiltered with a molecular weight cut off of 300,000 to remove free sugar chains and the tris (hydroxymethyl) aminomethane; and
  • step (D4) In the step (D3), including the step of replacing the buffer solution of the solution from which the free sugar chain and the tris (hydroxymethyl) aminomethane have been removed with a HEPES buffer solution (pH 7.2), The manufacturing method as described.
  • composition according to item 39 wherein the sugar chain of the sugar chain-modified ribosome is sialyle Lewis X group, and the sialyle Lewis X group has a modified binding density of 0.025 mg sugar chain Z mg lipid.
  • composition according to item 75 for delivering the substance to an inflamed site or cancer tissue.
  • item 76 for delivering the substance to an inflamed site or cancer tissue.
  • the inflammatory site or cancer tissue comprises a parenchyma.
  • a carrier for use in molecular imaging or in vivo imaging wherein the carrier comprises a sugar chain-modified ribosome according to items 1-37.
  • the sugar chain of the sugar chain-modified ribosome is a sialyl Lewis X group, and the sialyl Lewis X group is contained at a modified binding density of 0.025 mg sugar chain Zmg lipid, and the carrier is a labeling substance at an inflammatory site or cancer tissue.
  • a system for molecular or in vivo imaging of a site of interest comprising:
  • the label After a sufficient time for the label to accumulate at the target site, the presence or absence of the label in the living body is examined, and the function or structure of the living body is imaged by the label.
  • a system according to item 81, wherein the means for checking for the presence of the marker includes a scanning microscope (item 87).
  • a system according to item 86, wherein the means for checking the presence of the sign further comprises a stick objective lens.
  • a method for producing a fluorescent dye-containing sugar chain-modified ribosome according to Item 53 wherein: a) a step of providing a ribosome encapsulating fluorescence to which a linker protein is bound; b) a step of hydrophilizing the ribosome ; c) binding 3, 3, 1 dithiobis (sulfosuccinimidyl propionate) to the ribosome; and
  • Step c) and step b) are performed in order after step a).
  • the c) step is as follows:
  • the step b) includes the following:
  • the step d) includes the following:
  • (dl) a step of completely dissolving a desired sugar chain in purified water, and 1 to: preparing a sugar chain solution having a LOmM concentration; (d2) If necessary, ammonium bicarbonate (pH 7-14) is added to the aqueous sugar chain solution at a concentration of about 0.2-1 Og / mL, and 3-20 at 20-40 ° C. Stirring for 7 days, incubating at 2-8 ° C for 20-60 minutes and filtering through a filter to prepare an aminated sugar chain solution;
  • Item 91 The manufacturing method according to Item 90.
  • a kit for producing a fluorescent dye-containing sugar chain-modified ribosome A kit for producing a fluorescent dye-containing sugar chain-modified ribosome
  • V Means for binding the sugar chain to the ribosome
  • a kit comprising:
  • a kit for producing a fluorescent dye-containing sugar chain-modified ribosome comprising:
  • the present invention provides a sugar chain-modified ribosome useful for molecular imaging, a method for producing the same, and a method for using the same.
  • the sugar chain-modified ribosome of the present invention greatly expands the scope of development of a DDS preparation capable of providing a desired drug at a target delivery site. According to the present invention, it becomes possible to develop and put into practical use a delivery system necessary for realizing new therapies in various fields such as cancer therapy, gene therapy, and regenerative medicine.
  • Various sugar chain-modified ribosomes useful for such molecular imaging are provided for the first time by the present invention.
  • the present invention provides a method for producing a ribosome containing a labeled sugar chain. By this method, it is possible to label a desired sugar chain and examine the distribution and locality of the sugar chain molecule. Furthermore, it is possible to screen sugar chains for lectins that are specifically expressed in pathological tissues using a disease model.
  • FIG. 1 shows image data in cancer-bearing mice using cy5.5-encapsulated sugar chain-modified ribosomes or cy5.5-encapsulated ribosomes.
  • Top row before ribosome administration
  • left and center 2nd row immediately after K1-3 ribosome administration
  • left and center 3rd row 1 day after administration of K1-3 ribosome
  • left and center 4th row 2 days after administration of K1-3 ribosome
  • Right 2nd stage Immediately after administration of ribosome without sugar chain
  • Right 3rd stage 1 day after administration of ribosome without sugar chain
  • Right 4th stage 2 days after administration of ribosome without sugar chain.
  • the rightmost bar shows the fluorescence signal intensity obtained by imaging.
  • the upper red color of the bar is the lower color with the strongest signal. , Indicating that the fluorescent signal is weak.
  • the fluorescence signal becomes weaker as the white color above the bar becomes stronger and the lower it becomes.
  • FIG. 2 shows image data in a tumor-bearing mouse using cy5.5-encapsulated sugar chain-modified ribosome or cy5.5-encapsulated ribosome.
  • Top row before ribosome administration
  • left 2nd row immediately after K1 3 liposome administration
  • left 3rd row 8 hours after administration of K1-3 ribosome
  • left 4th row 1 day after administration of K1-3 liposome
  • right 2 Stage Immediately after administration of ribosomes without sugar chains
  • right Third stage 8 hours after administration of liposomes without sugar chains
  • Right fourth stage one day after administration of ribosomes without sugar chains.
  • the rightmost bar shows the fluorescence signal intensity obtained by imaging.
  • the red color at the top of the bar is the lower color with the strongest signal, indicating that the fluorescence signal is weaker.
  • the whiter above the bar the stronger the fluorescent signal, the lower the fluorescent signal, the weaker the fluorescent signal.
  • the unit is photon count, photon / second (ph / s): the number of fluorescence signals photon counted per second.
  • FIG. 3 shows image data in cancer-bearing mice using cy5.5-encapsulated sugar chain-modified ribosomes or cy5.5-encapsulated ribosomes.
  • Top row before ribosome administration
  • left 2nd row immediately after K1 3 liposome administration
  • left 3rd row 1 day after administration of K1-3 ribosome
  • 4th row 2 days after administration of K1-3 liposome
  • 5th left Eye 3 days after K1-3 ribosome administration
  • left 6th stage 4 days after administration of K1-3 ribosome
  • 2nd stage Immediately after administration of ribosome without sugar chain
  • right 3rd stage 1 day after administration of ribosome without sugar chain
  • right 4th stage 2 days after administration of ribosome without sugar chain
  • right 5th stage 3 days after administration of ribosome without sugar chain
  • 6th stage 4 days after administration of ribosome without sugar chain.
  • the rightmost bar shows the fluorescence signal intensity obtained by imaging.
  • the red color above the bar shows the lower color with the strongest signal, indicating that the fluorescence signal is weaker.
  • the black-and-white imaging diagram the whiter above the bar, the weaker the fluorescent signal, the weaker the fluorescent signal.
  • the unit is photon count, photon / second (ph / s): represents the number of fluorescence signals photon counted per second.
  • FIG. 4 shows image data in cancer-bearing mice using cy5.5-encapsulated sugar chain-modified ribosomes or cy5.5-encapsulated ribosomes.
  • Top row before ribosome administration
  • left second row K3—3 lipo
  • left 3rd stage K3-3 ribosome administration 1 day
  • left 4th stage K3-3 liposome administration 2 days
  • left 5th stage K3-3 ribosome administration 3 days
  • right 2nd stage Immediately after administration of ribosomes without sugar chains
  • right third stage 1 day after administration of ribosomes without sugar chains
  • right 5th stage 3 days after administration of ribosomes without sugar chains.
  • the rightmost bar shows the fluorescence signal intensity obtained by imaging.
  • the red color above the bar shows the lower color with the strongest signal, indicating that the fluorescence signal is weaker.
  • the black-and-white imaging diagram the whiter above the bar, the weaker the fluorescent signal, the weaker the fluorescent signal.
  • the unit is photon count, photon / second (ph / s): represents the number of fluorescence signals photon counted per second.
  • FIG. 5 shows image data in arthritic mice using cy5.5-encapsulated sugar chain-modified ribosomes or cy5.5-encapsulated ribosomes.
  • Top row before ribosome administration
  • left 2nd row immediately after K1-3 liposome administration
  • left 3rd row 1 day after administration of K1-3 ribosome
  • 4th row 2 days after administration of K1-3 liposome
  • left 5 Stage K1—3 days after administration of 3 ribosomes
  • right 2nd stage right after administration of liposome without sugar chain
  • Right 3rd stage 1 day after administration of ribosome without sugar chain
  • right 4th stage 2 days after administration of ribosome without sugar chain
  • Right 5th row 3 days after administration of ribosome without sugar chain.
  • the rightmost bar shows the fluorescence signal intensity obtained by imaging.
  • the red color above the bar is the lower color with the strongest signal, indicating that the fluorescence signal is weaker.
  • the fluorescence signal is weaker as the white color above the bar becomes stronger and the lower the bar.
  • the unit is photon count, photon / secon nd (ph / s): represents the number of fluorescent signal photon counted per second.
  • FIG. 6 shows image data in arthritic mice using cy5.5-encapsulated sugar chain-modified ribosomes or cy5.5-encapsulated ribosomes.
  • Top row before ribosome administration
  • left 2nd row immediately after K1-3 liposome administration
  • left 3rd row 1 day after administration of K1-3 ribosome
  • 4th row 2 days after administration of K1-3 liposome
  • right 2 Stage Immediately after administration of ribosomes without sugar chains
  • right Third stage 1 day after administration of liposomes without sugar chains
  • Right 4th stage 2 days after administration of ribosomes without sugar chains.
  • the rightmost bar shows the fluorescence signal intensity obtained by imaging.
  • the red color above the bar is the lower color with the strongest signal, indicating that the fluorescence signal is weaker.
  • the fluorescent signal becomes stronger as the white color above the bar becomes lower. It shows that the fluorescence signal is weak.
  • the unit is photon count, photon / secon nd (ph / s): represents the number of fluorescent signal photon counted per second.
  • FIG. 7 shows image data in arthritic mice using cy5.5-encapsulated sugar chain-modified ribosomes or cy5.5-encapsulated ribosomes. From the left, the ribosome group without sugar chain, the K1-3 liposome administered group, the K1-4 ribosome administered group, the K1-5 ribosome administered group, and the K1-6 ribosome administered group are shown. In each group, the upper part shows before ribosome administration, the middle part shows immediately after ribosome administration, and the lower part shows one day after ribosome administration. The rightmost bar shows the fluorescence signal intensity obtained by imaging. In the color imaging diagram, the red color at the top of the bar indicates the weaker the fluorescent signal the lower the color with the strongest signal.
  • the fluorescent signal is weaker as the white color above the bar becomes stronger and the fluorescent signal becomes lower.
  • the unit is photon count, photon / second (ph / s): The number of fluorescence signals photon counted per second.
  • FIG. 8 shows image data in arthritic mice using cy5.5-encapsulated sugar chain-modified ribosomes or cy5.5-encapsulated ribosomes. From the left, the ribosome group without sugar chain, the K3-3 liposome administration group, the K3-4 ribosome administration group, the K3-5 ribosome administration group, and the K3-6 ribosome administration group are shown. In each group, the upper part shows before ribosome administration, the middle part shows immediately after ribosome administration, and the lower part shows one day after ribosome administration. The rightmost bar shows the fluorescence signal intensity obtained by imaging. In the color imaging diagram, the red color at the top of the bar indicates the weaker the fluorescent signal the lower the color with the strongest signal.
  • the fluorescent signal is weaker as the white color above the bar becomes stronger and the fluorescent signal becomes lower.
  • the unit is photon count, photon / second (ph / s): The number of fluorescence signals photon counted per second.
  • FIG. 9 shows image data in arthritic mice using cy5.5-encapsulated sugar chain-modified ribosomes or cy5.5-encapsulated ribosomes. From the left, the ribosome group without sugar chain, the K2-3 liposome administration group, the K2-4 ribosome administration group, the K2-5 ribosome administration group, and the K2-6 ribosome administration group are shown. In each group, the upper part shows before ribosome administration, the middle part shows immediately after ribosome administration, and the lower part shows one day after ribosome administration. The rightmost bar shows the fluorescence signal intensity obtained by imaging. In the color imaging diagram, the red color above the bar is the most signal The stronger the lower color, the weaker the fluorescent signal.
  • the fluorescent signal is weaker as the white color above the bar becomes stronger and the fluorescent signal becomes lower.
  • the unit is photon count, photon / second (ph / s): The number of fluorescence signals photon counted per second.
  • FIG. 10 shows image data in arthritic mice using cy5.5-encapsulated sugar chain-modified ribosomes or cy5.5-encapsulated ribosomes.
  • arthritis mouse Z sugar chain-free ribosome group From left, arthritis mouse Z sugar chain-free ribosome group, arthritis mouse ZK1-3 ribosome administration group, arthritis mouse ZK1-4 ribosome administration group, normal mouse Z sugar chain-free ribosome administration group, normal mouse ZK1-4 ribosome administration group .
  • the upper row shows before ribosome administration
  • the second row shows immediately after ribosome administration
  • the third row shows one day after ribosome administration
  • the fourth row shows two days after ribosome administration.
  • the rightmost bar shows the fluorescence signal intensity obtained by imaging.
  • the color imaging diagram shows that the fluorescent signal is weaker as the upper red color of the cell becomes the lower color with the strongest signal.
  • the whiter the bar the stronger the fluorescent signal
  • the lower the bar the weaker the fluorescent signal.
  • the unit is photon count, photo n / second (ph / s): represents the number of fluorescent signal photon counted per second.
  • FIG. 11 shows image data in arthritic mice using cy5.5-encapsulated sugar chain-modified ribosomes or cy5.5-encapsulated ribosomes.
  • Top row before ribosome administration
  • left 2nd row immediately after K1-3 ribosome administration
  • left 3rd row 1 day after K1-3 ribosome administration
  • right 2nd row immediately after administration of liposomal without glycans
  • right 3rd row 1 day after administration of ribosome without sugar chain.
  • the rightmost bar shows the fluorescence signal intensity obtained by imaging. In the color imaging diagram, the red color at the top of the bar shows the lower color with the strongest signal, indicating that the fluorescence signal is weaker.
  • the unit is photon count, photon / second (ph / s): the number of fluorescence signals photon counted per second.
  • FIG. 12 shows image data in arthritic mice using cy5.5-encapsulated sugar chain-modified ribosomes or cy5.5-encapsulated ribosomes.
  • Top row before ribosome administration
  • left 2nd row immediately after K1—2 ribosome administration
  • left 3rd row 1 day after administration of K1—2 ribosome
  • right 2nd row immediately after administration of liposomal without glycans
  • right 3rd row 1 day after administration of ribosome without sugar chain.
  • the bar on the right is an image
  • the fluorescence signal intensity obtained by ging is shown. In the color imaging diagram, the red color at the top of the bar shows the lower color with the strongest signal, indicating that the fluorescence signal is weaker.
  • the unit is photon count, photon / second (ph / s): the number of fluorescence signals photon counted per second.
  • FIG. 13 shows image data in arthritic mice using cy5.5-encapsulated sugar chain-modified ribosomes or cy5.5-encapsulated ribosomes.
  • Top row before ribosome administration
  • left 2nd row immediately after administration of K3-2 ribosome
  • left 3rd row 1 day after administration of K3-2 ribosome
  • 2nd row immediately after administration of K3-4 liposome
  • middle 3rd row K3—4 days after ribosome administration
  • right 2nd row immediately after liposome without sugar chain
  • right 3rd row 1 day after administration of ribosome without sugar chain.
  • the rightmost bar shows the fluorescence signal intensity obtained by imaging.
  • the red color at the top of the bar shows the lower color with the strongest signal, indicating that the fluorescence signal is weaker.
  • the whiter the bar the stronger the fluorescent signal, and the lower the bar, the weaker the fluorescent signal.
  • the unit is photon count, photon / second (ph / s): the number of fluorescence signals photon counted per second.
  • FIG. 14 shows brain image data in normal mice using cy5.5-encapsulated sugar chain-modified ribosomes or cy5.5-encapsulated ribosomes.
  • Upper left 1 hour after administration of K1 ribosome, administration of ribosome without sugar chain from left side, administration of K1-3 ribosome, no administration
  • upper center 1 hour after administration of K2 liposome, administration of ribosome without sugar chain, administration of K2-3 Ribosome administration, not administered
  • upper right 1 hour after administration of K3 ribosome, ribosome without glycan from left side, K3-3 liposome administration, not administered
  • lower left 1 day after administration of K1 ribosome, left side force ribosome without sugar chain Administration
  • K1—3 ribosome administration, K1—4 ribosome administration, K1—6 ribosome administration not administered
  • middle lower K2 ribosome administration 1 day later, left side force also without glycans
  • the rightmost bar shows the fluorescence signal intensity obtained by imaging.
  • the red color above the bar is the lower color with the strongest signal, indicating that the fluorescence signal is weaker.
  • White In the black imaging diagram, the fluorescent signal is weaker as the fluorescent signal becomes stronger and lower as the white color above the bar.
  • the unit is photon count, photon / second (ph / s): the number of fluorescence signals photon counted per second.
  • FIG. 15 shows liver image data in normal mice using cy5.5-encapsulated sugar chain-modified ribosomes or cy5.5-encapsulated ribosomes.
  • Left top 1 hour after administration of K1 ribosome, administration of ribosome without sugar chain from the left side, administration of K1 3 ribosome, unadministration
  • center top 1 hour after administration of K2 ribosome, administration of ribosome without sugar chain on the left side
  • K2-3 ribosome Administration not administered
  • top right 1 hour after administration of K3 ribosome, left-side force without glycosome administration, administration of K3-3 ribosome, no administration
  • left 2nd 1 hour after administration of K1 ribosome, no glycan from left side Ribosome administration, K1-4 ribosome administration, K1-6 ribosome administration, middle 2nd stage: K2 ribosome administration 1 hour later, glycosome without glycans from the left side, K2-4 ribosome administration, K2-6 ribo
  • the rightmost bar shows the fluorescence signal intensity obtained by imaging.
  • the upper red color of the bar shows the strongest signal, and the lower the color, the weaker the fluorescent signal.
  • the fluorescence signal becomes weaker as the white color above the node becomes stronger and the lower it becomes.
  • FIG. 16 shows kidney image data in normal mice using cy5.5-encapsulated sugar chain-modified ribosomes or cy5.5-encapsulated ribosomes.
  • Left top 1 hour after administration of K1 ribosome, administration of ribosome without sugar chain from the left side, administration of K1 3 ribosome, unadministration
  • center top 1 hour after administration of K2 ribosome, administration of ribosome without sugar chain on the left side
  • K2-3 ribosome Administration not administered
  • top right 1 hour after administration of K3 ribosome, left-side force without glycosome administration, administration of K3-3 ribosome, no administration
  • left 2nd 1 hour after administration of K1 ribosome, no glycan from left side Ribosome administration, K1-4 ribosome administration, K1-6 ribosome administration, middle 2nd stage: K2 ribosome administration 1 hour later, glycosome without glycans from the left side, K2-4 ribosome administration, K2-6 ribo
  • the rightmost bar shows the fluorescence signal intensity obtained by imaging.
  • the upper red color of the bar shows the strongest signal, and the lower the color, the weaker the fluorescent signal.
  • the fluorescence signal becomes weaker as the white color above the node becomes stronger and the lower it becomes.
  • the unit is photon count, photon / second (ph / s): The number of fluorescence signals photon counted per second.
  • Figure 17 shows the use of cy5.5-encapsulated sugar chain-modified ribosome or cy5.5-encapsulated ribosome.
  • the image data of the spleen in a normal mouse are shown.
  • Left top 1 hour after administration of K1 ribosome, administration of ribosome without sugar chain from the left side, administration of K1 3 ribosome, unadministration
  • center top 1 hour after administration of K2 ribosome, administration of ribosome without sugar chain on the left side
  • K2-3 ribosome Administration not administered
  • top right 1 hour after administration of K3 ribosome, left-side force without glycosome administration, administration of K3-3 ribosome, no administration
  • left 2nd 1 hour after administration of K1 ribosome, no glycan from left side Ribosome administration, K1-4 ribosome administration, K1-6 ribosome administration, middle 2nd stage: K2 ribosome administration 1 hour later, glycosome without glycans from the left side, K2-4
  • the rightmost bar shows the fluorescence signal intensity obtained by imaging.
  • the upper red color of the bar shows the strongest signal, and the lower the color, the weaker the fluorescent signal.
  • the fluorescence signal becomes weaker as the white color above the node becomes stronger and the lower it becomes.
  • the unit is photon count, photon / second (ph / s): The number of fluorescence signals photon counted per second.
  • FIG. 18 shows lung image data in normal mice using cy5.5-encapsulated sugar chain-modified ribosomes or cy5.5-encapsulated ribosomes.
  • Upper left 1 hour after administration of K1 ribosome, ribosome without glycan from left side, administration of K1 3 ribosome, not administered
  • upper center K2 1 hour after administration of posomes, administration of ribosome without sugar chain, administration of K2-3 ribosome, not administered
  • top right 1 hour after administration of K3 ribosome, administration of ribosome without sugar chain, administration of K3-3 ribosome, administration of untreated .
  • Left 2nd 1 hour after administration of K1 ribosome, administration of ribosome without sugar chain from left side, administration of K1-4 ribosome, administration of K1-6 ribosome
  • middle 2nd stage 1 hour after administration of K2 liposome, left side force is also sugar Unchained ribosome administration, K2—4 ribosome administration,
  • the rightmost bar shows the fluorescence signal intensity obtained by imaging.
  • the red color at the top of the bar indicates the weaker the fluorescent signal the lower the color with the strongest signal.
  • the fluorescence signal is weaker as the white color above the bar becomes stronger and the lower the bar.
  • the unit is photon count, photon / second (ph / s): The number of fluorescence signals photon counted per second.
  • FIG. 19 shows spleen image data in normal mice using cy5.5-encapsulated sugar chain-modified ribosomes or cy5.5-encapsulated ribosomes.
  • Left top 1 hour after administration of K1 ribosome, administration of ribosome without sugar chain from the left side, administration of K1 3 ribosome, unadministration
  • center top 1 hour after administration of K2 ribosome, administration of ribosome without sugar chain on the left side
  • K2-3 ribosome Administration non-administration
  • top right 1 hour after administration of K3 ribosome, administration of ribosome without sugar chain on the left side, K3-3 ribosome administration, unadministered
  • left 2nd stage 1 hour after K1 ribosome administration, glycosome-free ribosome administration, K1-4 ribosome administration, K1-6 ribosome administration, middle 2nd stage: ⁇ 2 ribosome administration 1 After time, administration of ribosome without sugar chain from the left side, ⁇ 2-4
  • the rightmost bar shows the fluorescence signal intensity obtained by imaging.
  • the upper red color of the bar shows the strongest signal, and the lower the color, the weaker the fluorescent signal.
  • the fluorescence signal becomes weaker as the white color above the node becomes stronger and the lower it becomes.
  • the unit is photon count, photon / second (ph / s): The number of fluorescence signals photon counted per second.
  • FIG. 20 shows heart image data in normal mice using cy5.5-encapsulated sugar chain-modified ribosomes or cy5.5-encapsulated ribosomes.
  • Left top 1 hour after administration of K1 ribosome, administration of ribosome without sugar chain from the left side, administration of K1 3 ribosome, unadministration
  • center top 1 hour after administration of K2 ribosome, administration of ribosome without sugar chain on the left side
  • K2-3 ribosome Administration not administered
  • top right 1 hour after administration of K3 ribosome, left-side force without glycosome administration, administration of K3-3 ribosome, no administration
  • left 2nd 1 hour after administration of K1 ribosome, no glycan from left side Ribosome administration, K1-4 ribosome administration, K1-6 ribosome administration, middle 2nd stage: 1 hour after administration of K2 ribosome, administration of ribosome without sugar chain from the left side, ⁇ 2-4 ribosome administration, ⁇ 2
  • the rightmost bar shows the fluorescence signal intensity obtained by imaging.
  • the upper red color of the bar shows the strongest signal, and the lower the color, the weaker the fluorescent signal.
  • the fluorescence signal becomes weaker as the white color above the node becomes stronger and the lower it becomes.
  • the unit is photon count, photon / second (ph / s): The number of fluorescence signals photon counted per second.
  • FIG. 21 shows whole body image data other than the head in a normal mouse using cy5.5-encapsulated sugar chain-modified ribosome or cy5.5-encapsulated ribosome.
  • Upper row administration of ribosome without sugar chain, from the left side before administration, immediately after administration, 5 minutes after administration, 10 minutes after administration, 15 minutes after administration, 20 minutes after administration, 25 minutes after administration
  • lower row administration of K1-3 ribosome, From left to right before administration, immediately after administration, 5 minutes after administration, 10 minutes after administration, 15 minutes after administration, 20 minutes after administration, and 25 minutes after administration.
  • the rightmost bar shows the fluorescence signal intensity obtained by imaging.
  • the red color above the bar shows the lower color with the strongest signal, indicating that the fluorescence signal is weaker.
  • the whiter the bar the stronger the fluorescent signal, and the lower the bar, the weaker the fluorescent signal.
  • Unit is photon count, ph oton / second (ph / s): Represents the number of fluorescent signal photon counted per second.
  • FIG. 22 shows whole body image data other than the head in normal mice using cy5.5-encapsulated sugar chain-modified ribosomes or cy5.5-encapsulated ribosomes.
  • Top Ribosome without sugar chain, from left side, before administration, immediately after administration, 5 minutes after administration, 10 minutes after administration, 15 minutes after administration, 20 minutes after administration, 25 minutes after administration
  • 2nd stage K1-4 Ribosome administration, from left side, before administration, immediately after administration, 5 minutes after administration, 10 minutes after administration, 15 minutes after administration, 20 minutes after administration, 25 minutes after administration.
  • Third stage K16 ribosome administration, from left side, before administration, immediately after administration, 5 minutes after administration, 10 minutes after administration, 15 minutes after administration, 20 minutes after administration, and 25 minutes after administration.
  • the rightmost bar shows the fluorescence signal intensity obtained by imaging.
  • the red color at the top of the bar indicates the lower the color with the strongest signal, the weaker the fluorescent signal.
  • the fluorescent signal is weaker as the white color above the bar becomes stronger and the fluorescent signal becomes lower.
  • the unit is photon count, photon / second (ph / s): The number of fluorescence signals photon counted per second.
  • FIG. 23 shows whole body image data other than the head in a normal mouse using cy5.5-encapsulated sugar chain-modified ribosome or cy5.5-encapsulated ribosome.
  • Upper row ribosome without sugar chain, before administration, right after administration, 5 minutes after administration, 10 minutes after administration, 15 minutes after administration, 20 minutes after administration, 25 minutes after administration
  • lower administration of K2-3 ribosome, From left to right before administration, immediately after administration, 5 minutes after administration, 10 minutes after administration, 15 minutes after administration, 20 minutes after administration, and 25 minutes after administration.
  • the rightmost bar shows the fluorescence signal intensity obtained by imaging. In the color imaging diagram, the red color above the bar shows the lower color with the strongest signal, indicating that the fluorescence signal is weaker.
  • the unit is photon count, photo / second (ph / s): the number of photon photon counted per second.
  • FIG. 24 shows whole body image data other than the head in normal mice using cy5.5-encapsulated sugar chain-modified ribosome or cy5.5-encapsulated ribosome.
  • Top row glycoside without glycan administration, from left side, before administration, immediately after administration, 5 minutes after administration, 10 minutes after administration, 15 minutes after administration, 20 minutes after administration, 25 minutes after administration
  • 2nd step K2-4 Ribosome administration, from left side, before administration, immediately after administration, 5 minutes after administration, 10 minutes after administration, 15 minutes after administration, 20 minutes after administration, 25 minutes after administration.
  • Third stage K2— 6 Ribosome administration, before administration, immediately after administration, 5 minutes after administration, 10 minutes after administration, 15 minutes after administration, 20 minutes after administration, and 25 minutes after administration.
  • the rightmost bar shows the fluorescence signal intensity obtained by imaging.
  • the red color at the top of the bar indicates the lower the color with the strongest signal, the weaker the fluorescent signal.
  • the fluorescent signal is weaker as the white color above the bar becomes stronger and the fluorescent signal becomes lower.
  • the unit is photon count, photon / second (ph / s): The number of fluorescence signals photon counted per second.
  • FIG. 25 shows whole body image data other than the head in a normal mouse using cy5.5-encapsulated sugar chain-modified ribosome or cy5.5-encapsulated ribosome.
  • Upper row ribosome without sugar chain, before administration, right after administration, 5 minutes after administration, 10 minutes after administration, 15 minutes after administration, 20 minutes after administration, 25 minutes after administration
  • lower row administration of K3-3 ribosome, From left to right before administration, immediately after administration, 5 minutes after administration, 10 minutes after administration, 15 minutes after administration, 20 minutes after administration, and 25 minutes after administration.
  • the rightmost bar shows the fluorescence signal intensity obtained by imaging. In the color imaging diagram, the red color above the bar shows the lower color with the strongest signal, indicating that the fluorescence signal is weaker.
  • the unit is photon count, photo / second (ph / s): the number of photon photon counted per second.
  • FIG. 26 shows whole body image data other than the head in a normal mouse using cy5.5-encapsulated sugar chain-modified ribosome or cy5.5-encapsulated ribosome.
  • Top row glycosome without glycans administered, from left side before administration, immediately after administration, 5 minutes after administration, 10 minutes after administration, 15 minutes after administration, 20 minutes after administration, 25 minutes after administration
  • 2nd step K3-4 Ribosome administration, from left side, before administration, immediately after administration, 5 minutes after administration, 10 minutes after administration, 15 minutes after administration, 20 minutes after administration, 25 minutes after administration.
  • Second stage K3-6 ribosome administration, from left side, before administration, immediately after administration, 5 minutes after administration, 10 minutes after administration, 15 minutes after administration, 20 minutes after administration, and 25 minutes after administration.
  • the rightmost bar shows the fluorescence signal intensity obtained by imaging.
  • the red color at the top of the bar indicates the lower the color with the strongest signal, the weaker the fluorescent signal.
  • the fluorescent signal is weaker as the white color above the bar becomes stronger and the fluorescent signal becomes lower.
  • the unit is photon count, photon / second (ph / s): 1 second Represents the number of fluorescence signals photon counted.
  • FIG. 27 shows image data of the whole body other than the head in normal mice using cy5.5-encapsulated ribosomes. From left to right before administration, immediately after administration, 30 minutes later, 1 day later, 2 days later. The rightmost bar indicates the fluorescence signal intensity obtained by imaging. In the color imaging diagram, the red color at the top of the bar indicates the weaker the fluorescent signal the lower the color with the strongest signal. In the black and white imaging diagram, the whiter color above the bar indicates that the fluorescence signal is weaker as the fluorescent signal is stronger and lower.
  • the unit is photon count, photonZsecond (phZs): Fluorescent signal photon count per second
  • FIG. 28 shows whole body image data other than the head in normal mice using cy5.5-encapsulating sugar chain-modified ribosome (K1-3). From the left side, before administration, immediately after administration, 30 minutes later, 1 day later. The rightmost bar shows the fluorescence signal intensity obtained by imaging. The color image shows that the red color above the bar is the lower color with the strongest signal, the weaker the fluorescent signal. In the black-and-white imaging diagram, the whiter above the bar, the stronger the fluorescent signal, and the lower the bar, the weaker the fluorescent signal.
  • the unit is phot on count, photonZsecond (phZs): Counted per second: 3 ⁇ 4: Represents the number of photon photon.
  • FIG. 29 shows whole body image data other than the head in normal mice using cy5.5-encapsulated sugar chain-modified ribosome (K1-4). From left to right before administration, immediately after administration, 30 minutes later, 1 day later, 2 days later. The rightmost bar shows the fluorescence signal intensity obtained by imaging. In the power error imaging diagram, the red color above the bar indicates the weaker fluorescent signal, as the lower color is the strongest signal. In the black-and-white imaging diagram, the whiter above the bar, the stronger the fluorescent signal, and the lower the bar, the weaker the fluorescent signal.
  • the unit is photon count, photon / second (ph / s): The number of fluorescent signal photons counted per second.
  • FIG. 30 shows whole body image data other than the head in normal mice using cy5.5-encapsulating sugar chain-modified ribosome (K1-6). From left to right before administration, immediately after administration, 30 minutes later, 1 day later, 2 days later. The rightmost bar shows the fluorescence signal intensity obtained by imaging. Power In the Ler image, the fluorescent signal is weaker as the red color above the bar is the lower color with the strongest signal. In the black-and-white imaging diagram, the whiter above the bar, the stronger the fluorescent signal, and the lower the bar, the weaker the fluorescent signal. The unit is photon count, photon / second (ph / s): The number of fluorescent signal photons counted per second.
  • FIG. 31 shows a schematic diagram of a sugar chain-modified ribosome encapsulating a fluorescent substance.
  • FIG. 32 shows an example of a calibration curve for measuring the amount of ribosome protein.
  • FIG. 33 shows an example of a calibration curve for measuring the lipid content of ribosome.
  • FIG. 34 shows an example of particle size distribution of ribosome.
  • FIG. 35 shows an example of a fluorescent substance.
  • FIG. 36 shows an example of a fluorescent substance.
  • FIG. 37 shows an example of a fluorescent substance.
  • FIG. 38 shows an example of a fluorescent substance.
  • FIG. 39 shows an example of a fluorescent substance.
  • FIG. 40 shows a schematic diagram for the preparation of sugar chain-modified ribosomes.
  • Tris, DTSSP and SLX are the following abbreviations.
  • HSA human serum albumin
  • BS human serum albumin
  • Tris bis (sulfosuccinimidyl) suberate group
  • Tris tris (hydroxymethyl) a
  • Minomethane group DTSSP; 3, 3, monodithiopis (sulfosuccinimidyl propionate), S LX; Siaryl Lewis X group.
  • FIG. 41 shows the particle size distribution of SLX—Lipo—Cy5.5 and Lipo—Cy5.5.
  • the ribosome solution was diluted 50 times with distilled water.
  • Vertical axis relative intensity of dynamic light scattering (%), horizontal axis: particle size (logarithm: diameter (nm)).
  • Solid line SLX—Lipo—Cy5.5.
  • Dashed line Lipo-Cy5.5.
  • the particle size was measured by Zetasizer Nano—S90.
  • FIG. 42 shows the stability of SLX-Lipo-Cy5.5 after 6 hours storage at 4 ° C.
  • the liposome solution was diluted 50 times with distilled water.
  • Vertical axis relative intensity of dynamic light scattering (%), horizontal axis: particle size (logarithm: diameter (nm)).
  • Solid line Immediately after preparation, dashed line: after storage at 4 ° C for 6 hours. The particle size was measured from Zetasizer Nano—S90.
  • FIG. 43 shows ribosome accumulation in the inflamed area in rheumatoid arthritis mice.
  • the SLX—Lipo—Cy5.5 or Lipo—Cy5.5 was administered via the tail vein (50 lZ mouse).
  • Inflamed areas (back of the hind paw) of the same mice were observed before administration, 0 hours after injection, and 24 hours after injection. Measured with explore Optix (Ex: 680 nm, Em: 700 nm). The data is correct.
  • FIG. 44 shows the accumulation of various sugar chain-modified ribosomes in the inflammatory region.
  • Lipo-Cy5.5, SLX-Lipo-Cy5.5 and G4GN-Lipo-Cy5.5 were also administered via tail vein force (50; ⁇ mice).
  • the inflamed area (back of the hind paw) was observed 24 hours after injection.
  • G4 GN N-acetyl lactosamine.
  • FIG. 45 shows the relationship between the sugar chain density on the ribosome surface and accumulation in the inflamed area.
  • Li po -Cy5.5 or SLX—Lipo—Cy5.5 (D1-D5) was administered via the tail vein (50 1Z mice).
  • the inflamed area (back of the hind paw) was observed 24 hours after injection. Measured with explore Optix (Ex: 680 nm, Em: 700 nm).
  • the density of each sugar chain indicates the concentration ( ⁇ g / ml) of the reaction mixture when the sugar chain is bound to the liposome surface.
  • FIG. 46 shows the accumulation of ribosomes in blood vessels and surrounding tissues in the inflamed area.
  • SLX Lipo—Cy5.5 or Lipo—Cy5.5 (100 1 / mouse) was administered via the tail vein.
  • AO 0.5%, w / v%) (150 1) was administered via the tail vein immediately before observation. Measured with IV-100. Cy5.5 is shown in red (Ex: 633 nm, Em: 69 3 nm), and ataridin orange is shown in green (Ex: 488 nm, Em: 526 nm). Bar: 20 ⁇ mo Arrow: Blood vessel.
  • FIG. 47 shows the accumulation of SLX-Lipo-Cy5.5 in the tumor area of tumor-bearing mice.
  • S LX-Lipo -Cy5.5 or Lipo—Cy5.5 was administered via the tail vein (200 lZ mice).
  • the tumor area (right thigh area) of the same mouse was observed before administration, 0 hours, 24 hours, 48 hours, 72 hours and 96 hours after administration.
  • FIG. 48 shows the fluorescence distribution in the body 96 hours after injection.
  • SLX—Lipo—Cy5.5 was also administered to the tail vein force (200 1Z mice). The whole body was observed 96 hours after the injection. Measured with explore Optix (Ex: 680 nm, Em: 700 nm).
  • FIG. 49 shows the movement of fluorescence from blood vessels to surrounding tissues in the tumor region.
  • SL X—Lipo—Cy5.5 or Lipo—Cy5.5 was administered via the tail vein (100 lZ mice). Forty-eight hours after injection, blood vessels and surrounding tissues in the tumor area (right thigh area) were observed.
  • AO 0.5%, w / v%) (150 / z l) was administered via tail vein force immediately before observation. Measured with IV-100. Cy5.5 is shown in red (Ex: 633 nm, Em: 693 nm) and atalidine orange is shown in green (EX: 488 nm, Em: 526 nm). Bar: 20 m.
  • Solid arrow Blood vessel. Striped arrows: tumor tissue (tumor cells). Triangle: White blood cell.
  • FIG. 50 is an image of cancer-bearing mice administered with Cy7-encapsulating sugar chain-modified ribosome (K-1) taken over time using a fluorescence imaging device eXplore Optix (GE Healthcare). .
  • a fluorescence imaging device eXplore Optix GE Healthcare.
  • FIG. 51 is a photograph of a tumor site of a tumor-bearing mouse administered with Cy 3 encapsulated sugar chain-modified ribosome (K-1) using a fluorescence microscope CKX41 (OLYMPUS). Six hours after administration, the tumor-bearing mice to which Cy3-encapsulated sugar chain-modified ribosome (K-1) was administered were found to have higher fluorescence intensity in the tumor tissue than the control.
  • FIG. 52 shows the particle size distribution of ribosomes produced by the conventional method and the centrifugal method.
  • A Conventional method
  • B Centrifugal method.
  • FIG. 53 shows absorption spectra for ribosomes produced by the conventional method and the centrifugal method. Thick line: Conventional method, Thin line: Centrifugal method.
  • the “sugar chain” refers to a compound formed by one or more unit sugars (monosaccharide and Z or a derivative thereof). When two or more unit sugars are connected, each unit sugar is linked by dehydration condensation using a glycosidic bond.
  • sugar chains examples include polysaccharides contained in the living body (glucose, galactose, mannose, fucose, xylose, N-acetylethyldarcosamine, N-acetylethylgalatosamine, sialic acid and In addition to their conjugates and derivatives), there are a wide range of sugar chains that are degraded or derived from complex biomolecules such as degraded polysaccharides, glycoproteins, proteoglycans, glycosaminodaricans, glycolipids, etc. It is not limited to them. Therefore, in the present specification, the sugar chain can be used interchangeably with “polysaccharide”, “sugar”, and “carbohydrate”.
  • sugar chain may include both sugar chains and sugar chain-containing substances.
  • monosaccharides such as dalcoose, galactose, mannose, fucose, xylose, N-acetylyldarcosamine, N-acetylgalatatosamine, sialic acid and their complexes and derivatives
  • the human body is expected to have several hundreds of sugar chains with various structures, and there are several useful structures in the human body.
  • sugar chain group is a name given when a sugar chain is bonded to another group.
  • the sugar chain group refers to a monovalent or divalent group depending on the case.
  • examples of the sugar chain group include sialyl Lewis X group, N-acetylyl lactosamine group, and ⁇ 1-6 mannobiose group.
  • sugar or “monosaccharide” refers to polyhydroxyaldehyde or polyhydroxyketone containing at least one hydroxyl group and at least one aldehyde group or ketone group, and constitutes a basic unit of a sugar chain. To do.
  • sugar is also referred to as a carbohydrate, and both are used interchangeably.
  • a sugar chain refers to a chain or a sequence in which one or more sugars are linked, and when referred to as a sugar or a monosaccharide, it refers to one unit constituting the sugar chain.
  • Ser is usually classified as a lipid, but in the present specification, it is treated as a saccharide unless otherwise specified because it also falls within the definition of a kind of saccharide constituting a glycan.
  • Ser is usually classified as an amino acid.
  • ⁇ and j8 since it also falls within the definition of a kind of sugar constituting a sugar chain, it is treated as a sugar unless otherwise mentioned.
  • the two cyclic anomers are denoted by ⁇ and j8. It may be expressed as a or b for display reasons. Therefore, in the present specification, ⁇ and a, j8 and b are used interchangeably for the anomeric notation.
  • galactose refers to any isomer, but typically 13 D It is galactose and is used to refer to j8-D-galactose unless otherwise stated.
  • acetylylgalatatosamine refers to any isomer, but is typically N-acetylyl-a-D galactosamine, and unless otherwise specified, N-acetylyl-a-D galactosamine. Used as a pointer.
  • mannose refers to any isomer, but is typically a-D-mannose, and is used to refer to ex D-mannose unless otherwise specified.
  • glucose refers to any isomer, typically j8-D.
  • Glucose unless otherwise mentioned, is used to refer to 13 D-glucose.
  • acetylyldarcosamine refers to any isomer, but is typically N-acetylenic ⁇ D darcosamine, and unless otherwise specified, refers to ⁇ acetylene ⁇ -D-darcosamine. Used as a thing.
  • fucose refers to any isomer, but is typically a L-fucose, and is used to refer to ⁇ L-fucose unless otherwise specified.
  • acetyl-neuraminic acid refers to any isomer, but is typically ⁇ - ⁇ acetylneuraminic acid, and ⁇ - ⁇ -acetylenuraminic acid is referred to unless otherwise specified. Used as a pointer.
  • serine refers to any isomer, but is typically L-serine, and is used to refer to L-serine unless otherwise specified.
  • the reducing end of the sugar is aminated, and the ability to bind to other components such as albumin via the amino group.
  • the reducing end hydroxyl group Note that refers to those substituted with amine groups.
  • Monosaccharides are generally joined by glycosidic bonds to form disaccharides and polysaccharides.
  • the direction of the bond with respect to the plane of the ring is indicated by ⁇ and j8.
  • Also described are specific carbon atoms that form a bond between two carbons.
  • a monosaccharide is represented by an anomaly-like A monosaccharide.
  • the e-glycosidic bond between C-1 in galactose and C-4 in glucose is represented by Gal
  • Siaryl Lewis X (SLX) is represented as Neu5Ac a 2,3Gal ⁇ 1,4 (Fuc ⁇ 1,3) GlcNAc.
  • N-acetyllactosamine (G4GN) is represented as Gal jS 1, 4GlcNAc.
  • ⁇ 1-6 Mannobiose (A6) is expressed as Man a 1, 6Man.
  • Branches of sugar chains are represented by parentheses, and are arranged immediately to the left of the unit sugar to be bound. For example,
  • sugar chain used in the present specification examples include sugar chains selected from the group having sialyl Lewis X, N-acetyllactosamine, ⁇ 1-6 mannobiose, and combinations of two or more thereof. However, it is not limited to these. The reason why two or more combinations can be used is not limited by theory, but each of the sugar chains has specificity for a lectin localized in the tissue or cell of the intended delivery site. This is because even if they are mixed, it is thought that their uniqueness will be exhibited.
  • the “ribosome” usually means a closed vesicle composed of a lipid layer assembled in a film form and an inner aqueous layer.
  • phospholipids typically used it is possible to incorporate cholesterol, glycolipids, and the like. Since ribosomes are closed vesicles containing water inside, it is possible to retain water-soluble drugs and the like in the vesicles. Therefore, these ribosomes are used to deliver drugs and genes that cannot pass through the cell membrane into the cell. In addition, its biocompatibility is good, so it is highly expected as a nanoparticulate carrier material for DDS.
  • the ribosome imparts a structural unit having a functional group that imparts an ester bond (for example, a glycolipid, redesignoside, phosphatidylglycerol, etc.) or a peptide bond in order to attach a modifying group. It may have a structural unit having a functional group (for example, phosphatidylethanolamine).
  • Ribosome can be prepared by any method known in the art. For example, among them, a method using a cholic acid dialysis method is exemplified. In the cholic acid dialysis method, production is carried out by a) preparation of mixed micelles of lipid and surfactant, and b) dialysis of mixed micelles. Next, in a preferred embodiment of the sugar chain ribosome of the present invention, the coupling of a glycoprotein having a sugar chain bound to a protein for which it is preferable to use a protein as a linker to the ribosome is the following two-step reaction. Can be done by.
  • An example of the reaction flow is shown in Fig. 31.
  • a glycoprotein containing a desired sugar chain can be bound to a liposome, and a wide variety of glycoprotein 'liposome conjugates having the desired sugar chain can be obtained. It is very important to examine the particle size distribution to see the purity and stability of ribosomes. Examples of such methods include gel filtration chromatography (GPC), scanning electron microscopy (SEM), and dynamic light scattering (DLS).
  • Ribosomes of the molar ratio 35: 45: 5: 15 of dipalmitoyl phosphatidylcholine, cholesterol, dicetyl phosphate, contoside can be produced. This ribosome is stable even when stored at 4 ° C for several months.
  • the in vivo stability of ribosomes can be examined using mice. The ribosome is intravenously injected into the mouse, blood is collected after 3 hours, serum is prepared, and the ribosome is purified and collected by ultrafiltration using a membrane with a pore size of 0.03 m. As a result of the SEM observation, it can be confirmed that the ribosome morphology does not change even before and after the recovery for 3 hours in vivo.
  • Lipids constituting the sugar chain-modified ribosome of the present invention include, for example, phosphatidylcholines, phosphatidylethanolamines, phosphatidic acids, long-chain alkyl phosphates, glycolipids (gandariosides, etc.), phosphatidylglycerols. , Sphingomyelins, cholesterols and the like.
  • phosphatidylcholines include dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, and the like.
  • phosphatidylethanolamines include dimyristoyl phosphatidylethanol. And amine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine and the like.
  • Examples of phosphatidic acids include dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, and distearoyl phosphatidic acid.
  • Examples of long-chain alkyl phosphates include dicetyl phosphate.
  • glycolipids examples include galactosylceramide, darcosylceramide, latatosylceramide, phosphatide, globoside, and contosides.
  • Gandriosides include ganglioside GMl (Gal j8 1, 3GalNA C j 8 1, 4 (NeuA a 2, 3) Gal j8 1, 4Gl C j 8 1, 1, Cer), gandarioside GDla, gandarioside GTlb, etc. It is done.
  • phosphatidylglycerols dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol and the like are preferable.
  • phosphatidic acids, long-chain alkyl phosphates, glycolipids, and cholesterol have the effect of increasing the stability of ribosomes, so it is desirable to add them as constituent lipids.
  • phosphatidylcholines (molar ratio 0 to 70%), phosphatidylethanolamines (molar ratio 0 to 30%), phosphatidic acids, and long-chain alkyl phosphate groups
  • Power One or more lipids selected (molar ratio 0-30%)
  • glycolipids, phosphatidylglycerols and sphingomyelins also selected group power One or more lipids (molar ratio 0-40%)
  • those containing cholesterol miolar ratio 0 to 70%.
  • gandarioside glycolipid or phosphatidylglycerol. This is because the binding of a linker such as albumin becomes easy.
  • the ribosome according to the present invention contains gandarioside, glycolipid or phosphatidylglycerol, and can be linked with a linker such as a peptide to bind a sugar chain. .
  • the glycan-modified ribosome of the present invention containing the sugar chain contained in the glycolipid as a constituent component can be produced by combining gandarioside, glycolipid or phosphatidylglycerol. it can.
  • the ribosome in the present invention comprises phosphatidylethanolamine. By including phosphatidylethanolamine, it is easy to bond with a hydrophilic group (such as tris (hydroxyalkyl) aminoalkane).
  • the present invention provides a sugar chain-modified ribosome.
  • a sugar chain-modified ribosome Conventionally, in vivo, it has not been provided that sufficiently targets a desired target cell or tissue.
  • the present invention has the effect of enabling targeting that was impossible with conventional DDS materials by providing sugar chain-modified liposomes that are directed to desired target cells or tissues in the living body.
  • such a sugar chain-modified ribosome has a sugar chain having at least one structure selected from the group consisting of sialyl Lewis X, N-acetyllactosamine, ⁇ 1-6 mannobiose, and a combination force thereof. Are connected.
  • sugar chain-modified ribosome refers to a substance containing a sugar chain and a ribosome, and preferably a liposome modified by direct or indirect binding of sugar chains.
  • ribosomes One hour. Specifically describing the form of sugar chains bound to ribosomes,
  • the linker protein contained in the ribosome can bind to CH— ⁇
  • R 1 Linker protein group
  • R 2 Linker-protein cross-linking group
  • X and R 1 are CH—NH bonded, R 1 and R 2 are peptide bonded, and R 2 and R 3 are peptide
  • t can be represented by a structural formula.
  • the sugar chain-modified ribosome of the present invention is hydrophilicized by the following structure: Structure II Y-R 4 -R 5
  • Y a group in which the functional group b is removed from the structural unit force including the hydrophilic compound cross-linking group contained in the ribosome and the functional group b capable of peptide bonding
  • R 4 hydrophilic compound crosslinking group
  • R 5 hydrophilic compound group
  • Y and R 4 are peptide bonds
  • R 4 and R 5 are peptide bonds.
  • t can be represented by a structural formula.
  • the ribosome of the present invention has the above-described Structure I and Structure II, and is fluorescent.
  • the fluorescent property is that at least one of the components of the sugar chain-modified ribosome of the present invention has fluorescence, or the sugar chain-modified ribosome of the present invention has a new fluorescent property (for example, , A fluorescent dye).
  • Examples of the fluorescent element include, but are not limited to, a fluorescent dye, a fluorescent protein (eg, GFP, CFP, YFP, etc.), and a luminescent enzyme (eg, luciferase, etc.).
  • a fluorescent dye for example, cy5.5 (for example,
  • Alexa Fluor350 Alexa Fluor488, Alexa Fluor532, Alexa Fluor 546, Alexa Fluor555, Alexa Fluor 568 ⁇ Alexa Fluor 594 ⁇ Alexa Fluor633, Alexa
  • Forces including, but not limited to, Fluor 647, Alexa Fluor680, Alexa Fluor700, Alexa Fluor750, and FITC.
  • compatible with a ribosome is a substance that does not impair the stability of the ribosome when the substance is included in or bound to the ribosome.
  • the nature of Compatibility with ribosomes can be determined by measuring, for example, zeta potential, electromobility, particle size, lipid content, protein content, and the like.
  • the aggregation property of ribosome particles can be determined by the zeta potential.
  • the average particle size, the maximum range, the number of ribosomes in the maximum range, etc. can be analyzed based on the particle size and size distribution, and the homogeneity of the ribosome can be confirmed based on the analysis results.
  • Protein mass Lipid content: By measuring the ratio of protein content per lipid, it is possible to confirm whether the ribosome has an appropriate composition.
  • the sugar chain-modified ribosomes used herein can be included at a density suitable for delivery to the intended delivery site.
  • modified bond density is the amount of sugar chain used in producing a sugar chain-modified ribosome, and is the number of sugar chains bound per mg of lipid in the ribosome. Expressed as density (mg sugar chain Zmg lipid).
  • density mg sugar chain Zmg lipid.
  • the binding density of the sugar chain-modified ribosome of the present invention is not desired to be bound by theory, empirically, the amount of sugar chain used in the preparation is almost equal to the density of sugar chains bound to the ribosome. Proportionalness is a component. Therefore, in this specification, unless otherwise stated, the binding density is determined by the amount used at the time of preparation. In the in vitro mouth, for example, it can be determined indirectly using E-selectin.
  • the sugar chain-modified ribosome of the present invention can control the directivity to the target delivery site by selecting the type of sugar chain to be bound to the ribosome and the binding density.
  • Table 1 below shows ribosome numbers, sugar chain structures, and modified bond densities.
  • the preferred sugar chain-modified ribosome of the present invention as described in the above table can be produced by the following method. Specifically, this method comprises the steps of: (a) suspending lipids in methanol Z chloroform solution and stirring, evaporating the stirred solution, and drying the precipitate in vacuum to obtain a lipid membrane; b) Suspending the lipid membrane in a suspension buffer and sonicating; (c) mixing the sonicated solution with a fluorescent labeling solution to provide fluorescently labeled liposomes Step (d) Hydrophilic treatment of the ribosome with tris (hydroxyalkyl) aminoalkane; (e) Linker protein is bound to the hydrophilic ribosome and linked to the linker protein.
  • the fluorescent labeling solution in step (c) is 1, 1'-bis ( ⁇ -roxyruboxypentyl) 3, 3, 3 ', 3, -tetramethylindocarbocyanine 5, 5, -disulfonate potassium salt, di-hydroxy hydroxysuccinimide ester (cy5.5), the linker protein in step (e) is human serum albumin, and in step (f), the sugar chain and ribosome are Glycosylated liposomes are produced by conjugation under conditions suitable for delivery to the target delivery site.
  • ribosome and the linker, and the linker and the sugar chain are linked using a bifunctional cross-linking group (for example, 3, 3′-dithiopis (sulfosuccinimidyl propionate) (DTSSP)). It is preferable.
  • a bifunctional cross-linking group for example, 3, 3′-dithiopis (sulfosuccinimidyl propionate) (DTSSP)). It is preferable.
  • the sugar chain-modified ribosome of the present invention can encapsulate or bind a drug or gene.
  • the drugs include alkyl ⁇ anticancer agents, antimetabolites, plant-derived anticancer agents, anticancer antibiotics, biological response modifiers (BRM), cytodynamics, platinum complex anticancer agents, Immunotherapeutic agents, hormone anticancer agents, tumor drugs such as monoclonal antibodies, central nervous system drugs, peripheral nervous system, sensory organ drugs, respiratory disease drugs, cardiovascular drugs, digestive organ drugs, hormone drugs , Urogenital drugs, vitamins, nourishing tonics, metabolic drugs, antibiotics, chemotherapeutic drugs, testing drugs, anti-inflammatory drugs, eye disease drugs, central nervous system drugs, autoimmune drugs, cardiovascular system Drugs, lifestyle-related diseases such as diabetes, hyperlipidemia, corticosteroids, immunosuppressants, antibacterial agents, antiviral agents, angiogenesis inhibitors, cytoforce-in, chemokines, anti-site-forced antibodies, Chemokine antibodies, anti-cytokina, anti
  • linker is a molecule that mediates the binding between a sugar chain and the ribosome surface.
  • the sugar chain may be bound to the ribosome surface via a linker.
  • the linker can be appropriately selected by those skilled in the art, but those that are biocompatible are preferred, and are preferably pharmaceutically acceptable.
  • the “linker protein” refers to a protein, peptide, amino acid polymer of a linker molecule.
  • the linker protein used in the present specification can be, for example, a biological protein, preferably a human-derived protein, more preferably a human-derived serum protein, and still more preferably serum albumin. In particular, when human serum albumin is used, mice have a high uptake in each tissue.
  • the “linker group (protein) group” is a name given when the linker group (protein) is bound to another group.
  • the linker (protein) group is monovalent or divalent depending on the case. Examples thereof include a mammal-derived protein group, a human-derived protein group, a human serum protein group, and a serum albumin group.
  • the linker (protein) group is preferably derived from “human”. It is also the power that is considered highly compatible in human administration. Also, protein is preferred because it is not immunogenic!
  • the “crosslinking group” refers to a group that forms a chemical bond between molecules of a chain polymer so as to form a bridge. Typically, it acts between high molecules such as lipids, proteins, peptides, and sugar chains and other molecules (e.g., lipids, proteins, peptides, sugar chains), and is covalently linked within or between molecules. A group that forms a covalent bond that connects strong forces.
  • the crosslinking group varies depending on the target for crosslinking, and examples thereof include, but are not limited to, aldehydes (eg, dartal aldehyde), carpositimides, imide esters and the like. When the amino group-containing substance is crosslinked, an aldehyde-containing group such as dartal aldehyde can be used.
  • linker-protein cross-linking group refers to a group that forms a peptide bond between a ribosome and a sugar chain.
  • the linker protein cross-linking group varies depending on the target for cross-linking, such as bissulfosuccinimidyl suberate, disuccinimidyl glutarate, dithiobissuccinimidyl propionate, disuccinimidide.
  • Divalent reagents such as Rusberart, 3, 3, 1 dithiobis (sulfosuccinimidyl propionate), ethylene glycol bissuccinimidyl succinate, ethylene glycol bissulfosuccinimidyl succinate Etc. can be used.
  • linker protein crosslinking groups examples include 3, 3′-dithiopis (sulfosuccinimidyl). Propionate) group, bissulfosuccinimidyl suberate group, disuccinimidyl glutarate group, dithiobis succinimidyl propionate group, disuccinimidyl sulfate group, ethylene glycol bis succinimidyl group Mention may be made of a succinate group and an ethylene glycol bissulfosuccinimidyl succinate group.
  • the terms "protein”, “polypeptide”, “oligopeptide” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any length. .
  • the polymer may be linear or branched or cyclic.
  • the amino acid may be a modified amino acid, which may be natural or non-natural.
  • the term may also include those assembled into a complex of multiple polypeptide chains.
  • the term also encompasses natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, daricosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (eg, conjugation with a labeling component).
  • This definition also includes, for example, polypeptides containing one or more analogs of amino acids (eg, including non-natural amino acids, etc.), peptidomimetic compounds (eg, peptoids), and the art! Other modifications are included
  • protein refers to a polymer of amino acids having a relatively large molecular weight or a variant thereof, and when referring to “peptide”, it has a relatively small molecular weight. It should be understood that it may refer to a polymer of amino acids or variants thereof. Examples of such molecular weight include, but are not limited to, about 30 kDa, preferably about 20 kDa, more preferably about 10 kDa.
  • biologically derived protein refers to a protein derived from an organism, including any organism (eg, any type of multicellular organism (eg, animal (eg, Vertebrates, invertebrates), plants (eg monocotyledonous plants, dicotyledonous plants, etc.))).
  • organism eg, any type of multicellular organism (eg, animal (eg, Vertebrates, invertebrates), plants (eg monocotyledonous plants, dicotyledonous plants, etc.)).
  • the protein is derived from a vertebrate (for example, metaraunagi, shark eel, cartilaginous fish, teleost, amphibian, reptile, bird, mammal, etc.), more preferably a mammal (for example, a single hole, marsupial) , Rodents, skin wings, wings, carnivores, carnivores, long noses, odd hoofs, even hoofs, rodents, scales, sea cattle, cetaceans, primates , Rodents, maggots, etc.) are used. More preferably, a protein derived from a primate (eg, chimpanzee, second monkey, human) is used. Most preferably, a biological protein for administration is used. In this specification, when a biological protein shows a state of binding to another substance, it is called a biological protein group.
  • a biological protein group for example, metaraunagi, shark eel, cartilaginous fish, teleost, amphibian, reptile,
  • human-derived serum protein refers to a protein contained in a liquid portion that remains when human blood naturally coagulates.
  • a human-derived protein group when a human-derived protein shows a state of being bound to another substance, it is referred to as a human-derived protein group.
  • serum albumin refers to albumin contained in serum. In the present specification, when serum albumin shows a state of being bound to another substance, it is referred to as serum albumin group.
  • At least one of the ribosome membrane and the linker is hydrophilicized by binding a hydrophilic compound, preferably, a tris (hydroxyalkyl) aminoalkane. Moyo.
  • hydrophilization refers to binding of a hydrophilic compound to the ribosome surface.
  • the compound used for the hydrophilic property is a low molecular weight hydrophilic compound, preferably a low molecular weight hydrophilic compound having at least one OH group, more preferably a low molecular weight hydrophilic compound having at least two OH groups. Is mentioned. Further, a low molecular weight hydrophilic compound having at least one amino group, that is, a hydrophilic compound having at least one OH group and at least one amino group in the molecule.
  • the hydrophilic compound Since the hydrophilic compound is a small molecule, it does not hinder the progress of the sugar chain molecule recognition reaction by the lectin on the surface of the target cell membrane because it becomes a steric hindrance to the sugar chain.
  • the hydrophilic compound does not include a sugar chain to which a lectin used for directing a specific target such as a lectin can be bound in the sugar chain-modified ribosome of the present invention.
  • examples of such hydrophilic compounds include amino alcohols such as tris (hydroxyalkyl) aminoalkane including tris (hydroxymethyl) aminomethane, and more specifically, tris (hydroxy).
  • Droxymethinole) aminoethane tris (hydroxyethyl) aminoethane, tris (hydroxypropyl) aminoethane, tris (hydroxymethyl) aminomethane, tris (hydroxyethyl) Examples include aminomethane, tris (hydroxypropyl) aminomethane, tris (hydroxymethyl) aminopropan, tris (hydroxyethyl) aminopropane, and tris (hydroxypropyl) aminopropan.
  • alkyl refers to the loss of one hydrogen atom from an aliphatic hydrocarbon such as methane, ethane, or propane (herein “alkane” t). The resulting monovalent group, generally represented by CH 1 (where n is a positive integer). Alkyl is n 2n + l
  • substituted alkyl refers to an alkyl in which H of the alkyl is substituted with a substituent as defined below. Specific examples of these include C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, C1-C10 alkyl.
  • 1 to 11 alkyl or 1 to 12 alkyl C1-C2 substituted alkyl, C1-C3 substituted alkyl, C1-C4 substituted alkyl, C1-C5 substituted alkyl, C1-C6 Substituted alkyl, C1-C7 substituted alkyl, C1-C8 substituted alkyl, C1-C9 substituted alkyl, C1-C10 substituted alkyl, C1-C11 substituted alkyl or C1-C12 substituted Or alkyl.
  • alkanes these specific examples include C1-C2 alkanes, C1-C3 alkanes, C1-C4 alkanes, C1-C5 alkanes, C1-C6 alkanes, C1-C7 alkanes, C1-C8 alkanes, C1- C9 alkane, C1-C10 alkane, C1-C11 alkane or C1-C12 alkane, C1-C2-substituted alkane, C1-C3-substituted alkane, C1-C4-substituted alkane, C1-C5-substituted alkane C1-C6 substituted alkanes, C1-C7 substituted alkanes, C1-C8 substituted alkanes, C1-C9 substituted alkanes, C1-C10 substituted alkanes, C1-C11 substituted alkanes Or a C1-C12 substituted
  • C1-C10 substituted alkyl refers to C1-C10 alkyl, in which one or more hydrogen atoms are substituted with a substituent.
  • R C1-C6 alkyl is preferred, and C1-C6 alkyl is particularly preferred.
  • substituent R is present in one or more, and each independently represents hydrogen, alkyl, cycloalkyl. , Alcohol, cycloalkenyl, alkyl, alkoxy, carbocyclic group, heterocyclic group, halogen, hydroxy, thiol, silane-containing nitro, ami-containing carboxy, acyl, thiocarboxy, amide, substituted amide , Substituted carbo- yl, substituted thiocarbol, substituted sulfol, and substituted sulfiel.
  • a compound in which an amino group is introduced into a low molecular weight compound having an OH group can also be used as the hydrophilic compound of the present invention.
  • the compound is not limited, and examples thereof include a compound in which an amino group is introduced into a sugar chain without binding of a lectin such as cellopioose.
  • the ribosome surface is rendered hydrophilic using a divalent reagent for crosslinking and tris (hydroxymethyl) aminomethane on the lipid phosphatidylethanolamine of the ribosome membrane.
  • the general formula of the hydrophilic compound is represented by the following formula (1), formula (2), formula (3), and the like.
  • RR 3 and R 5 are C force C, preferably C to C, more preferably C force
  • z represents a reactive functional group that binds to the ribosomal lipid directly or to a divalent reagent for crosslinking, such as COOH, NH, NH, CHO, SH, NHS-ester, male
  • n represents a natural number.
  • the surface of the ribosome that has been rendered hydrophilic with such a hydrophilic compound is thinly covered with a hydrophilic compound.
  • the thickness of the cover of the hydrophilic compound is small, even when sugar chains are bound to ribosomes, the reactivity of sugar chains and the like cannot be suppressed.
  • hydrophilic compound group is a name when the above hydrophilic compound is bonded to another group.
  • the hydrophilic compound group can be monovalent or divalent depending on the case.
  • the "hydrophilic compound cross-linking group” is a group that is peptide-bonded to one chain protein and the other end is peptide-bonded to a sugar chain. A group that forms a peptide bond with a liposome or linker protein.
  • hydrophilic compound crosslinking groups include bis (sulfosuccinimidyl) suberate group, disuccinimidyl glutarate group, dithiobissuccinimidyl propionate group, disuccinimidyl suberate group, 3, 3, 1 dithiobis (sulfosuccinimidyl propionate) group, ethylene glycol bis succinimidyl succinate group and ethylene glycol bis sulfosuccinimidyl succinate group.
  • the hydrophilic compound crosslinking group is a bis (sulfosuccinimidyl) suberate group.
  • the hydrophilicity of the ribosome is a conventionally known method, for example, a method of producing a ribosome using a phospholipid obtained by covalently binding polyethylene glycol, polyvinyl alcohol, maleic anhydride copolymer or the like ( JP 2000-302685 (for example, CNDAC-containing ribosomal preparations dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine; dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol; N monomethoxypolyethyleneglycol succinyl distearoylphosphatidylethanolamine (hereinafter referred to as PEG2000—DSPE) having a molecular weight of about 2000 CNDAC hydrochloride, aqueous glucose solution and aqueous tre
  • the method using tris (hydroxymethyl) aminomethane of the present invention is preferable in several respects as compared with the conventional hydrophilic method using polyethylene glycol or the like.
  • tris (hydroxymethyl) aminomethane is a low molecular weight substance, so that conventional polyethylene glycol or the like can be used.
  • the ribosome according to the present invention has a good particle size distribution, component composition, and dispersion characteristics even after the hydrophilization treatment, and is excellent in long-term storage and in vivo stability. It is preferred for use in To make the ribosome surface hydrophilic using tris (hydroxymethyl) aminomethane, lipids such as dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, and distearoyl phosphatidylethanolamine are used.
  • Zipalmitoilho By binding a divalent reagent to a fat such as fatidylethanolamine, and then reacting tris (hydroxymethyl) aminomethane with one of the bonds of the divalent reagent, tris (hydroxymethyl) amino on the ribosome surface. Combine methane.
  • the ribosome obtained by hydrophilizing the ribosome is extremely stable in the living body, and the half life in the living body can be obtained without binding a sugar chain having a target directivity as described later. Since it has a long V, it can be suitably used as a drug carrier in a drug delivery system.
  • the present invention also includes a ribosome whose surface is hydrophilic with a low molecular weight compound.
  • delivery vehicle refers to a carrier (vehicle) that mediates delivery of a desired substance. If the substance to be delivered is a drug, it is referred to as a “drug delivery vehicle”.
  • Drug delivery system D rug Delivery System (DDS), also called drug delivery system, is sometimes classified into absorption-controlled DDS, controlled-release DDS, and target-oriented DDS.
  • the ideal DDS is a system that delivers a drug “to the necessary part of the body”, “a necessary amount”, and “for the required time”.
  • Targeting DDS (written as Targeting DDS, translated into target-oriented DDS) is categorized as noisy 'targeting (passive and target-oriented) DDS and active' targeting (active 'target-oriented) DDS.
  • the former is a method for controlling the behavior in the body using the physicochemical properties such as the particle size of the carrier (drug carrier) or hydrophilicity.
  • the latter is a method in which a special mechanism is added to these to actively control the direction to the target tissue.For example, it has a specific molecular recognition function for the target molecules of specific cells that constitute the target tissue.
  • a carrier to which an antibody or a sugar chain is bound and it is sometimes called a “missile drug”.
  • drug delivery vehicle refers to a vehicle for delivering a desired drug.
  • the present invention relates to a molecular imaging agent.
  • the molecular imaging agent can further include a pharmaceutically acceptable carrier and the like.
  • Pharmaceutically acceptable carriers include, for example, antioxidants, preservatives, colorants, flavors, and diluents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffering agents, delivery vehicles. , Diluents, excipients and Z or pharmaceutical adjuvants.
  • the molecular imaging agent of the present invention is administered in the form of a composition comprising a glycosylated ribosome together with one or more physiologically acceptable carriers, excipients or diluents.
  • a suitable vehicle can be water for injection, physiological solution, or artificial cerebrospinal fluid.
  • Acceptable carriers, excipients or stabilizers used herein are non-toxic to the recipient and are preferably inert at the dosages and concentrations used.
  • Such non-toxic and inert carriers include, for example, phosphate, citrate, or other organic acids; ascorbic acid, ⁇ -tocopherol; low molecular weight polypeptides; proteins (eg, serum albumin Hydrophilic polymers (eg, polyvinylpyrrolidone); amino acids (eg, glycine, glutamine, asnolaggin, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (Including glucose, mannose, or dextrin); chelating agents (eg, EDTA); sugar alcohols (eg, mannitol or sorbitol); salt-forming counterions (eg, sodium); and Z or nonionic surface activation Examples include, but are not limited to, agents such as Tween, pluronic or polyethylene glycol (PEG), polyethylene
  • Exemplary suitable carriers include neutral buffered saline or saline mixed with serum albumin.
  • the product is formulated as a lyophilizer using a suitable excipient (eg, sucrose).
  • suitable excipient eg, sucrose
  • Other standard carriers, diluents and excipients may be included as desired.
  • Other exemplary compositions comprise tris (hydroxymethyl) aminomethane buffer at pH 7.0-8.5 or acetate buffer at pH 4.0-5.5, which further comprises sorbitol or suitable Alternatives can be included.
  • the present invention provides a molecular imaging agent containing a sugar chain-modified ribosome.
  • the drug delivery vehicle of the present invention is a sugar chain having at least one structure selected from the group consisting of Gal, GalNAc, Man, Glc, GlcNAc, Fuc and Neu5Ac, preferably a sugar chain shown in Table 1 above.
  • Including a sugar chain-modified ribosome linked with The sugar chain-modified liposome may encapsulate or bind a drug or gene.
  • the term "molecular imaging agent” refers to a drug or factor used for imaging a function or structure of a living body. Examples thereof include those used for imaging cancer tissues and inflamed sites in vivo.
  • the molecular imaging agent of the present invention is used for administering a biological factor to a subject in need of a biological factor, and for the respiratory system, circulatory system, digestive system, urinary organ, genital system, It can also be used to treat mammals with central or peripheral nervous system disorders.
  • the molecular imaging agent of the present invention can also enhance the absorption controllability in the intestinal tract by adjusting the type of sugar chain and the binding density of the sugar chain-modified ribosome.
  • the sugar chain may be a silyl Lewis X group. This sialyl Lewis X group can be included in the sugar chain-modified ribosome with a modified bond density of 0.
  • sugar chain modified ribosomes are included at a modified bond density of 0.0025 mg sugar chain Z mg lipid to 0.1 mg sugar chain Z mg lipid (preferably 0.025 mg sugar chain / mg lipid). It can be done.
  • sugar chain-modified ribosome it is preferably included in the sugar chain-modified ribosome at a modified bond density of 0.0025 mg sugar chain Z mg lipid to 0.1 mg sugar chain Z mg lipid (preferably 0.025 mg sugar chain Z mg lipid). obtain.
  • the sugar chain may be an N-acetylyl lactosamine group.
  • the N-acetyllactosamine group can be contained in the sugar chain-modified ribosome with a modified binding density of 0. OOOlmg sugar chain Zmg lipid to 500 mg sugar chain Zmg lipid, preferably 0.025 mg sugar chain Zmg lipid.
  • it is preferably included in the sugar chain-modified ribosome at a modified bond density of 0.0025 mg sugar chain Z mg lipid to 0.1 mg sugar chain Z mg lipid (preferably 0.025 mg sugar chain / mg lipid).
  • the glycosylation ribosome has a modified binding density of 0.0025 mg sugar chain Zmg lipid to 0.5 mg sugar chain Zmg lipid (preferably 0.1 mg sugar chain / mg lipid).
  • the sugar chain may be an a 1-6 mannobiose group.
  • This ⁇ 1-6 mannobiose group can be contained in a sugar chain-modified ribosome with a modified bond density of 0. OOOlmg sugar chain Z mg lipid to 500 mg sugar chain / mg lipid.
  • it is preferably included in the glycosylated liposome at a modified bond density of 0.0025 mg sugar chain Zmg lipid to 0.1 mg sugar chain Zmg lipid (preferably 0.025 mg sugar chain Zmg lipid). obtain.
  • the imaging agent of the present invention can be used to image an inflammatory site, cancer tissue, cerebral blood vessel, liver or the like. These organizations may contain substance.
  • the molecular imaging agent of the present invention can be easily prepared by those skilled in the art by considering pH, isotonicity, stability, and the like.
  • the molecular imaging agent of the present invention is blended with a pharmaceutically acceptable carrier, and is a solid preparation such as a tablet, capsule, granule, powder, powder, etc.
  • Oral administration as a liquid preparation such as syrup, suspension, solution and the like.
  • the molecular imaging agent of the present invention is a physiologically acceptable carrier, excipient or stabilizer as necessary (Japanese Pharmacopoeia 14th edition or its latest edition, Remington's Phar maceuticai sciences, 18th Edition, AR Gennaro, ed., Mack Publishing Company, 1990, etc.) and a glycan composition having the desired degree of purity, and prepared in the form of a lyophilized cake or aqueous solution. Can be preserved.
  • the fluorescent dye-containing sugar chain-modified ribosome of the present invention enables imaging with higher sensitivity than conventional imaging agents. Because it can be distinguished from autofluorescence derived from biological components by selecting fluorescent dyes with excitation and fluorescence detection wavelengths of 500-700 nm long wavelength, it is possible to realize highly sensitive imaging from outside the living body. This is possible.
  • the amount of the molecular imaging agent used in the treatment method of the present invention depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, cell morphology or type. It can be easily determined by those skilled in the art in consideration of the above.
  • the frequency with which the treatment method of the present invention is applied to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, gender, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art. Examples of the frequency include administration once a few months every day (for example, once a week—once a month). 1 week—preferably given once a month, over time.
  • Molecular imaging agents can be formulated using pharmaceutically acceptable carriers well known in the art in dosage forms suitable for administration. Such carriers allow drug delivery vehicles to be formulated into liquids, gels, syrups, slurries, suspensions, etc. suitable for consumption by the patient.
  • the present invention provides a carrier for use in molecular or in vivo imaging.
  • This carrier may include a sugar chain-modified ribosome.
  • the sugar chain has a silyl Lewis X group. It can be.
  • This Siaryl Lewis X group can be included in the sugar chain-modified ribosome at a modified bond density of 0. OOOlmg sugar chain Zmg lipid to 500 mg sugar chain Zmg lipid.
  • it can be included in the glycan-modified ribosome with a modified binding density of preferably 0.0025 mg glycan Zmg lipid to 0.1 mg glycan Zmg lipid (preferably 0.025 mg glycan / mg lipid) .
  • the sugar chain-modified ribosome When imaging cancer tissue, it is preferably contained in the sugar chain-modified ribosome at a modified bond density of 0.0025 mg sugar chain Z mg lipid to 0.1 mg mg sugar chain Z mg lipid (preferably 0.025 mg sugar chain Z mg lipid). obtain.
  • the sugar chain may be an N-acetyllactosamine group.
  • the N-acetylyllactosamine group may be contained in the sugar chain-modified ribosome at a modified bond density of 0. OOOlmg sugar chain Zmg lipid to 5 OOmg sugar chain Zmg lipid, preferably 0.025 mg sugar chain Zmg lipid.
  • a modified bond density 0. OOOlmg sugar chain Zmg lipid to 5 OOmg sugar chain Zmg lipid, preferably 0.025 mg sugar chain Zmg lipid.
  • the sugar chain modified ribosome can preferably be included at a modified bond density of 0.0025 mg sugar chain Zmg lipid to 0.5 mg sugar chain Zmg lipid (preferably 0.1 mg sugar chain Zmg lipid).
  • the sugar chain may be a 1-6 mannobiose group.
  • This ⁇ 1-6 mannobiose group can be contained in a sugar chain-modified ribosome with a modified bond density of 0. OOOlmg sugar chain Zmg lipid to 500 mg sugar chain Zmg lipid.
  • it is preferably contained in the sugar chain-modified ribosome at a modified bond density of 0.0025 mg sugar chain Z mg lipid to 0.1 mg sugar chain Z mg lipid (preferably 0.025 mg sugar chain Z mg lipid). obtain.
  • the imaging agent of the present invention can be used to image an inflammatory site, cancer tissue, cerebral blood vessel, liver or the like. These organizations may contain substance.
  • the medium or composition of the present invention includes a composition in which the fluorescent dye, drug or biological agent is contained in the sugar chain-modified ribosome in an amount effective to achieve the intended purpose.
  • ⁇ Amount effective to treat '' is a term well recognized by those skilled in the art and refers to the amount of drug effective to produce the intended pharmacological result (e.g., prevention, treatment, prevention of recurrence).
  • a therapeutically effective amount is an amount sufficient to reduce the symptoms of the disease to be treated. is there.
  • One useful assay to ascertain an effective amount (eg, a therapeutically effective amount) for a given application is to measure the extent of recovery of the target disease.
  • the amount actually administered will depend on the individual to whom the treatment is to be applied, and is preferably an amount optimized to achieve the desired effect without significant side effects.
  • the determination of an effective dose is well within the ability of those skilled in the art.
  • Therapeutically effective doses, prophylactically effective doses, and the like and toxicity are standard pharmaceutical procedures in cell cultures or laboratory animals (e.g., ED, doses therapeutically effective in 50% of the population; and
  • the dose ratio between fruit and toxic effects is the therapeutic index, expressed as the ratio ED ZLD.
  • Drug delivery vehicles that exhibit large therapeutic indices are preferred.
  • Cell culture and animal experimentation power obtained Data used to formulate a range of quantities for human use.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED with little or no toxicity. This dosage is the dosage form used,
  • the dose is appropriately selected depending on age and other patient conditions, the type of disease, the type of cells used, and the like.
  • composition, molecular imaging agent, medium and the like of the present invention can be produced in a manner similar to a manner known in the art (for example, mixing, dissolution, etc.).
  • the composition of the present invention for delivering a substance to a desired site may contain a sugar chain-modified ribosome modified with a sugar chain.
  • the sugar chain may be a sialyl Lewis X group. This sialyl Lewis X group can be contained in sugar chain-modified ribosomes with a modified bond density of 0. OOOlmg sugar chain Zmg lipid to 500 mg sugar chain / mg lipid.
  • the sugar chain-modified ribosome can preferably be included at a modified bond density of 0.0025 mg sugar chain Zmg lipid to 0.1 mg sugar chain Zmg lipid (preferably 0.025 mg sugar chain Zmg lipid).
  • the sugar chain-modified ribosome contained in the composition of the present invention has a sugar chain of N-acetyllactosami. It can be a group.
  • the N-acetylyllactosamine group may be contained in the sugar chain-modified ribosome at a modified bond density of 0. OOOlmg sugar chain Zmg lipid to 5 OOmg sugar chain Zmg lipid, preferably 0.025 mg sugar chain Zmg lipid.
  • When imaging cerebral blood vessels preferably included in the sugar chain-modified ribosome with a modified bond density of 0.002 mg mg sugar chain Zmg lipid to 0.1 mg sugar chain Zmg lipid (preferably 0.025 mg sugar chain Zmg lipid).
  • the sugar chain modified ribosome can preferably be included at a modified bond density of 0.0025 mg sugar chain Zmg lipid to 0.5 mg sugar chain Zmg lipid (preferably 0.1 mg sugar chain Zmg lipid). .
  • the sugar chain may be an a 1-6 mannobiose group.
  • This ⁇ 1-6 mannobiose group can be contained in a sugar chain-modified ribosome with a modified bond density of 0. OOOlmg sugar chain Zmg lipid to 500 mg sugar chain Zmg lipid.
  • it is preferably contained in the sugar chain-modified ribosome at a modified bond density of 0.0025 mg sugar chain Z mg lipid to 0.1 mg sugar chain Z mg lipid (preferably 0.025 mg sugar chain Z mg lipid). obtain.
  • the imaging agent of the present invention can be used to image an inflammatory site, cancer tissue, cerebral blood vessel, liver or the like. These organizations may contain substance.
  • the "instruction” refers to a method for administering the sugar chain-modified ribosome of the present invention or a molecular imaging agent, etc. To obtain).
  • This instruction manual describes a word for instructing a procedure for administering the sugar chain-modified liposome or the molecular imaging agent of the present invention.
  • This instruction is prepared according to the format prescribed by the national regulatory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States) in the country where the present invention is implemented. It will be clearly stated that it has been approved.
  • the instructions are so-called package inserts and are usually provided in paper media, but are not limited thereto, for example, electronic media (for example, homepage (website) provided on the Internet, electronic (Child mail) can also be provided.
  • subject refers to an organism to which the treatment of the present invention is applied, and is also referred to as "patient”.
  • patient refers to an organism to which the treatment of the present invention is applied, and is also referred to as “patient”.
  • patient or subject may preferably be a human.
  • the present invention relates to a glycan for the manufacture of a medicament for treating disorders of the respiratory system, circulatory system, digestive system, urinary 'genital system, central nervous system, or peripheral nervous system.
  • a glycan for the manufacture of a medicament for treating disorders of the respiratory system, circulatory system, digestive system, urinary 'genital system, central nervous system, or peripheral nervous system.
  • modified ribosomes for treating disorders of the respiratory system, circulatory system, digestive system, urinary 'genital system, central nervous system, or peripheral nervous system.
  • any form described in the above can be used.
  • the present invention can also be used as a medicament.
  • the drugs encapsulated or bound to the sugar chain-modified ribosome of the present invention include, but are not limited to, for example, the following: alkylated anticancer agents, antimetabolites, plant-derived anticancer agents, Anticancer antibiotics, BRM, cytodynamics, platinum complex anticancer drugs, immunotherapy drugs, hormone anticancer drugs, tumor drugs such as monoclonal antibodies, central nervous system drugs, peripheral nervous system • sensory organ drugs, breathing Remedies for genital diseases, drugs for the cardiovascular system, drugs for the digestive organs, drugs for the hormonal system, drugs for the urinary organs, drugs for vitamins, nourishing tonics, metabolic drugs, antibiotics, chemotherapeutic drugs, testing drugs, anti-inflammatory Drugs, eye disease drugs, central nervous system drugs, self-immune drugs, cardiovascular drugs, lifestyle diseases such as diabetes and hyperlipidemia, corticosteroids, immunosuppressants, antibacterial drugs, antivirals
  • the present invention provides a method for imaging the respiratory system, circulatory system, digestive system, urinary 'genital system, central nervous system, or peripheral nervous system.
  • This method includes the step of administering a molecular imaging agent to a subject, the molecular imaging agent comprising a sugar chain-modified ribosome and a fluorescent dye, wherein the sugar chain-modified ribosome contains a sufficient amount of the fluorescent dye for detection.
  • a molecular imaging agent comprising a sugar chain-modified ribosome and a fluorescent dye, wherein the sugar chain-modified ribosome contains a sufficient amount of the fluorescent dye for detection.
  • the sugar chain-modified ribosome any form described in the above-mentioned (sugar chain-modified liposome) can be used.
  • the present invention provides a method for imaging inflammation or cancer.
  • This method comprises the step of administering a molecular imaging agent to a subject, the molecular imaging agent comprising a sugar chain-modified ribosome and a fluorescent dye, wherein the sugar chain-modified ribosome is Contains a sufficient amount of fluorescent dye for detection.
  • the sugar chain-modified ribosome any form described in the above (Sugar chain-modified ribosome) can be used.
  • the present invention provides a system for molecular imaging or in vivo imaging of a site of interest.
  • “molecular imaging” or “in vivo imaging” refers to imaging a function or structure of a living body.
  • the system for molecular imaging or in vivo imaging of the present invention is
  • the label includes, but is not limited to, a fluorescent substance, a radioactive substance, a coloring substance (for example, ⁇ gal), an issuing substance (for example, luciferase), and the like.
  • the sugar chain of the sugar chain-modified ribosome that can be used in the system of the present invention can be a silyl Lewis X group.
  • This sialyl Lewis X group can be included in a sugar chain-modified ribosome with a modified bond density of 0. OOOlmg sugar chain Z mg lipid to 500 mg sugar chain / mg lipid.
  • it is preferably contained in the sugar chain-modified ribosome at a modified bond density of 0.0025 mg sugar chain Z mg lipid to 0.1 mg sugar chain Z mg lipid (preferably 0.025 mg sugar chain / mg lipid).
  • the sugar chain modified ribosome may preferably be included at a modified binding density of 0.0025 mg sugar chain Z mg lipid to 0.1 mg sugar chain Z mg lipid (preferably 0.025 mg sugar chain Z mg lipid).
  • the sugar chain of the sugar chain-modified ribosome that can be used in the system of the present invention may be an N-acetyllactosamine group.
  • This N-acetyllactosamine group can be included in the sugar chain-modified ribosome at a modified binding density of 0. OOOlmg sugar chain / mg lipid to 500 mg sugar chain Z mg lipid, preferably 0.025 mg sugar chain Z mg lipid.
  • 0.0025 mg sugar chain Z mg lipid to 0.1 mg sugar chain Z mg lipid preferably 0.025 mg sugar It can be included in sugar chain modified ribosomes with a modified bond density of (chain Zmg lipid).
  • the sugar chain modified ribosome may preferably be included at a modified bond density of 0.0025 mg sugar chain Zmg lipid to 0.5 mg sugar chain Zmg lipid (preferably 0.1 mg sugar chain Zmg lipid).
  • the sugar chain of the sugar chain-modified ribosome that can be used in the system of the present invention may be a 1-6 mannobiose group.
  • This ⁇ 1-6 mannobiose group can be contained in the sugar chain-modified ribosome with a modified bond density of 0. OOOlmg sugar chain Zmg lipid to 500 mg sugar chain / mg lipid.
  • it is preferably contained in the sugar chain-modified ribosome at a modified bond density of 0.0025 mg sugar chain Z mg lipid to 0.1 mg mg sugar chain Z mg lipid (preferably 0.025 mg sugar chain Z mg lipid). obtain.
  • an inflamed site, cancer tissue, cerebral blood vessel, liver or the like can be imaged. These tissues may contain parenchyma.
  • the means for examining the presence or absence of the label used in the system of the present invention may be a scanning microscope.
  • the means for checking the presence or absence of the label further comprises a stick objective lens.
  • any substance for example, fluorescence, radiation, chromogenic substance (for example, ⁇ gal), issuing substance (for example, luciferase), etc.
  • Etc. This is because the function or structure of the living body can be imaged by detecting the label.
  • the present invention provides a method for treating a subject having a respiratory, circulatory, gastrointestinal, urinary 'genital, central or peripheral nervous system disorder.
  • This method comprises administering to a subject a molecular imaging agent for treating a disorder, the molecular imaging agent comprising a glycosylated ribosome and a pharmaceutically acceptable carrier, wherein the glycosylated ribosome comprises Contains an effective amount of the drug to treat the disorder.
  • a molecular imaging agent for treating a disorder the molecular imaging agent comprising a glycosylated ribosome and a pharmaceutically acceptable carrier, wherein the glycosylated ribosome comprises Contains an effective amount of the drug to treat the disorder.
  • the sugar chain-modified ribosome any form described in the above (Sugar chain-modified ribosome) can be used.
  • the present invention provides a method for treating a subject having inflammation or cancer.
  • This method comprises a molecular imaging agent for treating a disorder in a subject.
  • the molecular imaging agent comprises a glycosylated ribosome and a pharmaceutically acceptable carrier, and the glycosylated ribosome comprises an amount of an agent effective to treat the disorder.
  • the sugar chain-modified ribosome any form described in the above-mentioned (Sugar chain-modified ribosome) can be used.
  • the present invention provides a method for delivering a biological agent to a target site in a subject in need thereof.
  • This method includes the step of administering the sugar chain-modified ribosome of the present invention, wherein the sugar chain-modified ribosome contains an effective amount of the biological factor.
  • the sugar chain-modified ribosome any form described in the above (sugar chain-modified liposome) can be used.
  • the sugar chain-modified ribosome any form described in the above (Sugar chain-modified ribosome) can be used.
  • the present invention provides a method for producing a fluorescent dye-containing sugar chain-modified ribosome.
  • This production method includes: A) a step of forming a ribosome in which a fluorescent dye is encapsulated or bound; B) a step of hydrophilizing the ribosome; C) a step of binding the liposome to a linker protein. And D) a step of binding a sugar chain to the ribosome.
  • the sugar chain-modified ribosome any form described in the above-mentioned (Sugar chain-modified ribosome) can be used.
  • the present invention provides a method for producing a sugar chain-modified ribosome.
  • a lipid is suspended in methanol Z chloroform solution and stirred, the stirred solution is evaporated, and a precipitate is vacuum dried to obtain a lipid membrane; Suspending the lipid membrane in a suspension buffer and sonicating; (c) mixing the sonicated solution with a fluorescent labeling solution to provide fluorescently labeled ribosomes; (d) ) Hydrophilic treatment of the liposome with tris (hydroxyalkyl) aminoalkane; (e) Linker protein is bound to the hydrophile-treated ribosome to form a linker protein-bound ribosome.
  • the present invention provides a method for producing a sugar chain-modified ribosome for delivering a substance (eg, fluorescent dye, drug) to a target delivery site.
  • a substance eg, fluorescent dye, drug
  • This method comprises the steps of: (a) providing fluorescently labeled sugar chain-modified ribosomes having various sugar chain densities to achieve delivery to the target delivery site, comprising: (i) lipids Suspending in methanol Z chloroform solution and stirring, evaporating the stirred solution and drying the precipitate in vacuo to obtain a lipid membrane; (ii) the lipid membrane in suspension buffer Suspending and sonicating; (iii) mixing the sonicated solution with a fluorescent labeling solution; (b) glycan density on the glycan-modified ribosome; Determining the density to achieve optimal delivery to the delivery site; and (c) incorporating the substance (e.g., fluorescent dye, pharmaceutical) into the determined optimal glycosylated ribosome to contain the drug Including the step of generating ribosomes.
  • Incorporation of substances can include, for example, encapsulation, binding to the outer surface, and the like.
  • substances eg, fluorescent dyes, pharmaceuticals
  • sugar chain-modified ribosome any form described in the above (Sugar chain-modified ribosome) can be used.
  • any of those used to bind or encapsulate drugs in ribosomes is used.
  • DPPC dinormitoylphosphatidylcholine
  • DCP dicetylphosphate
  • DPPE dipalmitoylphosphatidylethanolamine
  • sodium cholate are weighed, Suspend in Tanol 'black mouth form solution (1: 1) and stir for 1 hour at 37 ° C; (2) Kuro mouth form' methanol is evaporated on a rotary evaporator and vacuum dried.
  • the ribosome itself can be produced according to a known method. Examples thereof include a thin film method, a reverse layer evaporation method, an ethanol injection method, and a dehydration-one rehydration method. [0153] It is also possible to adjust the particle size of ribosomes by using an ultrasonic irradiation method, an etrusion method, a French press method, a homogenization method, or the like.
  • the production method of the liposome itself of the present invention will be specifically described. For example, first, phosphatidylcholines, cholesterol, phosphatidylethanolamines, phosphatidic acids, gandariosides, glycolipids or phosphatidylglycerols are used as the ingredients.
  • lipid and surfactant sodium cholate Prepare mixed micelles of lipid and surfactant sodium cholate.
  • the combination of phosphatidic acids or long-chain alkyl phosphates such as dicetyl phosphate is essential to negatively charge the ribosome
  • the combination of phosphatidylethanolamines is a hydrophilic reaction site.
  • Formulation of favorosides or glycolipids or phosphatidylglycerols is essential as a binding site for the linker.
  • Group power consisting of redesignosides, glycolipids, phosphatidylglycerols, sphingomyelins, and cholesterols At least one selected lipid assembles in the ribosome and functions as a scaffold (raft) that binds the linker.
  • the ribosome of the present invention is further stabilized by the formation of rafts that can bind such proteins. That is, the liposome of the present invention has at least one lipid raft selected from the group power of gandarioside, glycolipid, phosphatidylglycerols, sphingomyelins and cholesterols for binding the linker. Contains the formed ribosome. And the ribosome is produced by carrying out ultrafiltration of the mixed micelle obtained by this.
  • the ribosome used in the present invention is a force that can be used even if it is a normal one.
  • the surface should be hydrophilic. After preparing the ribosome as described above, the ribosome surface is made hydrophilic.
  • the present invention also includes liposome itself by binding sugar chains that have been hydrophilicized using the above-mentioned hydrophilic compound.
  • hydrophilic ribosomes have the advantage that the stability of the ribosome itself is enhanced, and that the sugar chain is recognizable when the sugar chain is bound.
  • the ribosome of the present invention includes, for example, ribosomal constituent lipid strengths phosphatidylcholines (molar ratio 0 to 70%), phosphatidylethanolamines (molar ratio 0 to 30%), phosphatidic acids, long-chain alkyl phosphates and dicetyl.
  • Phosphate power group power selected One or more lipids selected (molar ratio 0-30%), gandariosides, glycolipids, phosphatases Tidyglycerols and Sphingomyelins Powerful group power Liposomes containing one or more selected lipids (molar ratio 0-40%) and cholesterols (molar ratio 0-70%).
  • the present invention further includes a method of rendering the ribosome hydrophilic by binding the above-described hydrophilic compound to the ribosome. It also includes hydrophilic liposomes with no sugar chains attached.
  • the target-directed ribosome or intestinal absorbable ribosome of the present invention can be produced by binding a sugar chain to a ribosome to which no sugar chain is bound.
  • the hydrophilic property can be performed as follows. (1) Carbonate buffer (50mM
  • the hydrophilic property can be carried out as follows. (1) To exchange the buffer with carbonate buffer (CB S buffer: 50 mM NaHCO 157 mM NaCl (pH 8.5)),
  • the production method of the present invention comprises: a) providing a ribosome encapsulating fluorescence to which a linker protein is bound; b) subjecting the ribosome to a hydrophilic treatment. C) a step of binding 3,3,1 dithiobis (sulfosuccimid-midylpropionate) to the ribosome; and d) binding a sugar chain to the linker protein in the ribosome, Including the step of generating chain-modified ribosomes, wherein the steps b) to c) can be performed in any order.
  • This embodiment further includes the step of e) hydrophilicizing the fluorescent dye-containing sugar chain-modified ribosome; f) filtering the solution containing the hydrophilic fluorescent dye-containing sugar chain-modified ribosome. obtain.
  • the sugar chain-modified ribosome any form described in the above-mentioned (Sugar chain-modified ribosome) can be used.
  • the production method of the present invention can be carried out by performing the step c) and the step b) in order after the step a).
  • the production method of the present invention is a powder wherein the step c) comprises (cl) a cross-linking agent (eg, 3, 3, -dithiobis (sulfosucci-midylpropionate)). Adding a solution A containing carbonate buffer to the body A to dissolve and preparing a mixed solution; and (c2) adding the mixed solution to the solution containing liposomes at room temperature for 16 to 20 hours Stirring, ultrafiltering with a molecular weight cut off of 30,000, desalting, and preparing a solution containing a fluorescently encapsulated ribosome bound with 3,3,1 dithiobis (sulfosuccinimidylpropionate).
  • a cross-linking agent eg, 3, 3, -dithiobis (sulfosucci-midylpropionate)
  • (Dl) a step of completely dissolving a desired sugar chain in purified water, and preparing a sugar chain solution having a concentration of 1 to LOmM (preferably 5 mM); (d2) if necessary, the sugar chain Add hydrogen carbonate (pH 7-14) to the aqueous solution at a concentration of about 0.2 to 1. Og / mL (preferably 0.6 g / mL), and 20 to 40 ° C.
  • the step e) includes (el) concentrating the solution containing the fluorescent dye-containing sugar chain-modified ribosome, and 2- [4- (2-hydroxyethyl) -1piperaduryl] ethanesulfonic acid.
  • Add solution C containing (HEPES) buffer, ultrafilter with a fraction of 30,000, concentrate, and contain the concentrated fluorescent dye-containing glycosylated liposomes Liquid was added to the C solution, the fluorescence-contained ribosomes sugar chains are bound may include the step of parent aqueous I spoon.
  • the step of preparing the aminated sugar chain solution of (d 2) can be omitted.
  • the buffer for dissolving the sugar chain may be any buffer as long as it does not have a primary amino group.
  • Refrigeration refers to a temperature in the range of about 1-12 ° C, preferably about 2-8 ° C, because the sugar chain is dissolved in a buffer containing a primary amino group, which prevents the sugar chain from binding to the ribosome.
  • the Tris buffer used in the present method is a buffer solution using Tris hydroxymethylammonium as a base component, for example, N tris (hydroxymethyl) 3-a.
  • a minopropanesulfonic acid buffer or the like can be used.
  • the sugar chains that can be used in the present method include, but are not limited to, sialic Lewis X, N-acetylyl lactosamine, ⁇ 1-6 mannobiose, and the like.
  • the sugar chain may preferably be Cialyl Lewis X.
  • the sugar chain that can be used by the method of the present invention can be a sugar chain capable of glycosylation reaction.
  • the amount of sugar chain added can range from about 1 to 250 L per mL of ribosome solution. Preferably, it can be about 2.5-125 / zL per mL of ribosome solution.
  • the sugar chain can be preferably added at a final concentration of 5 mM.
  • the present invention provides a kit for producing a fluorescent dye-containing sugar chain-modified ribosome.
  • This kit includes i) a force for encapsulating or binding a fluorescent dye (eg, cy5.5, cy3, cy7, etc.) into a liposome; ii) a hydrophilizing agent for the ribosome; iii) a linker for the ribosome A protein; and iv) a sugar chain; V) a means for binding the sugar chain to the ribosome.
  • a fluorescent dye eg, cy5.5, cy3, cy7, etc.
  • the present invention provides a kit for producing a fluorescent dye-containing sugar chain-modified ribosome.
  • This kit consists of (A) a solution containing a ribosome bound to a linker protein; (B) a powder A containing a cross-linking agent (eg, 3,3′-dithiobis (sulfosucci-midylpropionate), etc.) (C) Solution A containing carbonate buffer; (D) Solution B containing Tris buffer; and (E) Solution C containing HEPES buffer.
  • a cross-linking agent eg, 3,3′-dithiobis (sulfosucci-midylpropionate), etc.
  • C Solution A containing carbonate buffer
  • D Solution B containing Tris buffer
  • E Solution C containing HEPES buffer.
  • the sugar chain-modified ribosome any form described in the above (sugar chain-modified ribosome) can be used.
  • Tris buffer used in this kit is a buffer using Tris-hydroxyme thylammonium as a basic component, for example, N tris (hydroxymethyl) -3-amino.
  • Tris-hydroxyme thylammonium as a basic component
  • Propane sulfonate buffer or the like can be used.
  • “refrigerated (lower)” refers to a temperature ranging from about 1 ° C to about 12 ° C, preferably from about 2 ° C to about 8 ° C.
  • room temperature refers to a temperature in the range of about 15 ° C. to about 30 ° C., preferably about 20 ° C. to about 25 ° C.
  • the sugar chain that can be used in the sugar chain-modified ribosome of the present invention can be synthesized by a general sugar chain synthesis method. These methods include (1) chemical synthesis, (2) fermentation using genetically modified cells or microorganisms, (3) synthesis using a sugar hydrolase (glycosidase), (4) sugar transfer Examples of the synthesis method include an enzyme (glycosyltransferase).
  • a general sugar chain synthesis method include (1) chemical synthesis, (2) fermentation using genetically modified cells or microorganisms, (3) synthesis using a sugar hydrolase (glycosidase), (4) sugar transfer Examples of the synthesis method include an enzyme (glycosyltransferase).
  • the sugar chain used in the sugar chain-modified ribosome of the present invention may be a sugar chain synthesized by the above method or a commercially available sugar chain. [0170] (Sugar chain binding to ribosome)
  • any of the above-mentioned sugar chains may be directly bound to the ribosome prepared as described above, and further, a sugar chain may be bound via a linker.
  • the type of sugar chain to be bound to the ribosome is not limited to one, and a plurality of sugar chains may be bound.
  • the plurality of sugar chains may be a plurality of sugar chains having binding activity to different lectins existing in common on the cell surface of the same tissue or organ, and cells of different tissues or organs may be used. It may be a sugar chain having binding activity for different lectins present on the surface.
  • the sugar chain is mixed as a glycolipid to produce a ribosome, and the sugar chain is bound to the phosphosome of the ribosome after production and the sugar chain density is controlled.
  • a protein derived from a living body particularly a human-derived protein.
  • the protein derived from the living body is not limited, and examples include proteins existing in blood such as albumin, and other physiologically active substances existing in the living body.
  • animal serum albumin such as human serum albumin (HSA) and ushi serum albumin (BSA) to be raised, especially when human serum albumin is used, it has been shown by experiments on mice that there is a large uptake in each tissue. It has been confirmed.
  • the ribosome of the present invention is very stable, and can be subjected to post-treatments such as binding a protein, binding a linker, or binding a sugar chain after forming the ribosome. Therefore, a large amount of ribosome After the production, various ribosomes can be produced according to the purpose by binding different proteins according to the purpose or by linking a linker or sugar chain.
  • a sugar chain is directly bonded to a lipid constituting the ribosome via a linker.
  • the ribosome of the present invention has a complex carbohydrate type ligand such as glycolipid and glycoprotein, and is hydrophilized with a low molecular weight compound! It is a ribosome.
  • the liposome needs to contain a compound having a pharmaceutical effect.
  • the compound having a medicinal effect may be encapsulated in a ribosome or bound to the ribosome surface, but a protein having a medicinal effect may be used as a linker.
  • the protein may also serve as a linker for binding the ribosome and sugar chain and a protein having a medicinal effect.
  • the medicinal protein include physiologically active proteins.
  • a crosslinking group can be used when the ribosome and the linker are bound.
  • Ribosome NalO, Pb (0 CCH
  • Gandarioside present on the ribosome membrane surface is treated with an oxidizing agent such as NaBiO.
  • Gandarioside is coupled by a reductive amination reaction.
  • This linker is also preferably made hydrophilic, by binding a compound having a hydroxy group to Ringer protein, for example, bissulfosuccinimidyl suberate, disuccinimidyl glutarate.
  • a divalent reagent such as glycol bissulfosuccinimidyl succinate can be used to bind the above-mentioned hydrophilic compound such as tris (hydroxymethyl) aminomethane to a linker on the ribosome.
  • one end of a divalent reagent for crosslinking is bonded to all the amino groups of the linker.
  • a glycosylamine compound obtained by glycosylation of the reducing ends of various sugar chains is prepared, and the divalent cross-linked divalent conjugated glycans and amino groups of the sugar chain are bonded to each other as described above. Combine the other unreacted end of the reagent portion.
  • the covalent bond between the sugar chain and Z or hydrophilic compound and the ribosome, or the covalent bond between the sugar chain and Z or hydrophilic compound and the linker occurs when the ribosome is taken into the cell. It can also be cut.
  • a linker and a sugar chain are covalently bonded via a disulfide bond
  • the sugar chain is cleaved by reduction in the cell.
  • the surface of the liposome becomes hydrophobic, binds to the biological membrane, disturbs the membrane stability, and releases the drug contained in the ribosome.
  • the linker is bound to the ribosome as follows. (1) Add sodium metaperiodate / N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) and stir overnight under refrigeration. To do. (2) Remove free sodium metaperiodate and perform ultrafiltration (molecular weight cut off: 300,000) to exchange the buffer with PBS buffer (pH 8.0). (3) To the solution that had been added with HSAZPBS buffer (pH 8.0) and reacted at room temperature for 2 hours, sodium cyanoborate ZPBS buffer (pH 8.0) was added, and after 2 hours at room temperature, Stir overnight under refrigeration. (4) Free cyanoboron Perform ultrafiltration (molecular weight cut off: 300,000) to remove sodium acid and HSA, and exchange the buffer with carbonate buffer ( PH 8.5). As a result, the linker can be bound to the ribosome.
  • the linker is bound to the ribosome as follows. (1) Add sodium metaperiodate / N tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) and stir overnight under refrigeration to acidify the ribosome particle surface. . (2) Remove free sodium metaperiodate and perform ultrafiltration (molecular weight cut off: 300,000) to exchange the buffer with PBS buffer (pH 8.0). (3) After adding HSAZPBS buffer (pH 8.0) and reacting at room temperature for 2 hours, stir overnight under refrigeration. (4) Perform ultrafiltration (molecular weight cut off: 300, 000) for the purpose of removing free HSA and exchanging the buffer with carbonate buffer (PH8.5). As a result, the linker can be bound to the ribosome.
  • the linker is bound to the ribosome as follows. (1) Add sodium metaperiodate ZN tris (hydroxymethyl) 3-aminopropanesulfonic acid buffer (PH8.4) and stir overnight under refrigeration to acidify the ribosome particle surface. (2) To remove free sodium metaperiodate and replace the buffer with PBS buffer (pH 8.0), concentrate to 1/5 to 1/10 times the volume by ultrafiltration. Add pH 8.0 to the original volume. Repeat this operation twice. (3) After adding HSAZPBS buffer (pH 8.0) and reacting at room temperature for 2 hours, add sodium cyanoboronate ZPBS buffer (pH 8.0), and after 2 hours at room temperature, further refrigerate. Stir overnight under.
  • PBS buffer pH 8.0
  • the linker is bound to the ribosome as follows. (1) Add sodium metaperiodate ZN tris (hydroxymethyl) 3-aminopropanesulfonic acid buffer (PH8.4) and stir overnight under refrigeration to acidify the ribosome particle surface. (2) To remove free sodium metaperiodate and replace the buffer with PBS buffer (pH 8.0), concentrate to 1/5 to 1/10 times the volume by ultrafiltration. Add pH 8.0) To the capacity of Repeat this operation twice. (3) Add HSAZPBS buffer (pH 8.0), react at room temperature for 2 hours, and stir overnight under refrigeration.
  • PBS buffer pH 8.0
  • the sugar chain is bound to a linker on the ribosome.
  • the reducing end of the sugar constituting the sugar chain is glycosylated using ammonia salts such as NH HCO and NH COONH.
  • the sugar chain can be bound to the linker on the ribosome as follows. (1) Dissolve the sugar chain in purified water and react at 37 ° C for 3 days under ammonium hydrogen carbonate saturation. (Aminated sugar chain solution). (2) Add the crosslinking agent DTSSP (PIERCE) to the ribosome solution, and after 2 hours at room temperature, stir overnight under refrigeration. (3) Perform ultrafiltration (molecular weight cut off: 300,000) to remove free DTSSP. (4) Add aminated sugar chain solution and react at room temperature for 2 hours, then add tris (hydroxymethyl) aminomethane / carbonate buffer (pH 8.5) and stir overnight under refrigeration.
  • DTSSP crosslinking agent
  • Ribosome formation and fluorescence (eg, cy5.5, Cy3, Cy7, etc.) labeled HSA inclusion (step A); ribosome hydrophilic treatment (step B); ribosome-HSA binding (step C) and Finishing can be accomplished by 0.45 m filter filtration following glycosylation of liposomes (step D).
  • the amount of protein of the ribosome or sugar chain-modified ribosome of the present invention can be measured, for example, by the BCA method by measuring the amount of HSA encapsulated in the ribosome and the total amount of HSA coupled to the ribosome surface.
  • Micro BCA Protein Assay Reagent Kit Cat. No. 23235BN (PIERCE Co. LTD)
  • 2 mg Zml albumin BSA
  • the protein amount of the sugar chain-modified ribosome of the present invention can be, for example, in the range of 0.1 to: LmgZml.
  • the protein amount of the sugar chain-modified ribosome labeled with Cy3 can be, for example, 0.24 mg / ml or more.
  • Sugar-modified ribosomal protein labeled with Cy5.5 The mass can be, for example, 0.45 mgZml or more.
  • the protein amount of the sugar chain-modified liposome labeled with Cy7 can be, for example, 0.20 mgZml or more.
  • the amount of constituent lipids of the ribosome and sugar chain-modified ribosome of the present invention can be calculated, for example, by quantifying the amount of cholesterol.
  • FC Free cholesterol
  • the Detamina TC555 kit (catalog number UCCZEAN12 8) (KYOWA Co. LTD) can be used.
  • dilute the standard substance (50mgZml: cholesterol) with PBS buffer, 0, 0.1, 0.25, 0.5, 0.75, 1, 5, 10 g / 20 1 Prepare the solution.
  • the conversion formula for obtaining the lipid content is also expressed as follows, for example.
  • Lipid content ⁇ 50 / ⁇ 1) Cholesterol content 8 ⁇ 50 / ⁇ 1) ⁇ 4.51 (Conversion factor)
  • the ratio of protein to lipid in the ribosome can also lead to the results of protein quantification and lipid quantification described above, for example .
  • the sugar chain-modified ribosome of the present invention preferably has a ratio of protein to lipid of about 0.1 to about 0.5.
  • the lipid amount of the sugar chain-modified ribosome of the present invention can be, for example, in the range of 1 to 4 mgZml.
  • the amount of lipid of the sugar chain-modified ribosome labeled with Cy3 can be, for example, 1.2 mgZml or more.
  • the amount of lipid of the sugar chain-modified ribosome labeled with Cy5.5 can be, for example, 1.4 mgZml or more.
  • the amount of lipid of the sugar chain-modified ribosome labeled with Cy7 can be, for example, 2. lmgZml or more.
  • the particle size distribution and particle size of the ribosome and sugar chain-modified ribosome of the present invention can be determined by, for example, diluting ribosome particles 50-fold with purified water to produce a Zetasizer Nano (Nan-ZS: MAL VERN Co. LTD). Can be measured. An example of particle size distribution is shown in Fig. 34.
  • the ribosome and sugar chain-modified ribosome of the present invention preferably have a particle size of about 80 ⁇ m to about 165 nm in the maximum range of the particle size distribution. This is because a particle size of about 80 nm to about 165 nm can avoid the recognition of immune system cells such as macrophages, and can avoid the uptake of liver and spleen endothelial reticuloendothelial (RES) force to some extent.
  • RES liver and spleen endothelial reticuloendothelial
  • a sugar chain-modified ribosome having a particle size of about 80 nm to about 165 nm is suitable for encapsulating a drug and delivering the drug to a target organ or a diseased part.
  • the ribosome and sugar chain-modified ribosome of the present invention has an average particle size of about 50 nm to about 300 nm, preferably about 65 nm to about 165 nm, and more preferably about lOO nm. This is because if the particle size of the ribosome is too large, it enters the cell's endothelial system in the liver's spleen non-specifically, and if the particle size is too large, it tends to be phagocytosed by immune system cells such as macrophages. Moreover, it is desirable that the ribosome of the present invention is negatively charged. By being negatively charged, the interaction with negatively charged cells in the living body can be prevented.
  • the zeta potential of the ribosome surface of the present invention is 37 ° C in physiological saline. 50 to: LOmV, preferably 40 to 0 mV, more preferably 1 to 30 to 10 mV.
  • the zeta potential on the ribosome surface is not limited to forces that can be 120 mV to 30 mV at 25 ° C. Preferably, it is less than ⁇ 30 mV at 25 ° C.
  • the zeta potential on the ribosome surface may be less than ⁇ 120 mV (25 ° C.) or greater than 30 mV. This is because aggregation between ribosomes only has to occur.
  • Examples of the drug to be included in the sugar chain-modified ribosome of the present invention include alkyl-type anticancer agents, antimetabolites, plant-derived anticancer agents, anticancer antibiotics, BRM, cytodynamic ins, platinum complex anticancer agents, and immunotherapeutic agents.
  • Hormone anticancer agents tumor drugs such as monoclonal antibodies, central nervous system drugs, peripheral nervous system, sensory organ drugs, respiratory disease drugs, cardiovascular drugs, digestive organ drugs, hormone drugs, urogenital genitalia Drugs, vitamins' nourishing tonics, metabolic drugs, antibiotics, chemotherapeutic drugs, test drugs, anti-inflammatory drugs, eye disease drugs, central nervous system drugs, autoimmune drugs, cardiovascular drugs, diabetes, high Life-style related diseases such as lipemia, corticosteroids, immunosuppressants, antibacterial agents, antiviral agents, angiogenesis inhibitors, cytoforce-in, chemokines, anti-site force-in antibodies, anti-chemokine antibodies Anti-site power In'chemokine receptor antibody, siRNA, miRNA, smRNA, antisense Gene therapy-related nucleic acid preparation such as ODN or DNA, neuroprotective factor, antibody drug, molecular target drug, osteoporosis / bone metabolism improving drug, Examples include neuropeptides, bioactive peptides and proteins.
  • a tumor drug such as nitrogen mustard hydrochloride N-oxide, cyclophosphamide, ifosfamide, prusphan, hydrochloride-mustine, mitoblonitol, melphalan, dacarbazine, ramustine, estramustine phosphate sodium, etc.
  • Anti-metabolites such as alkylating agents, mercaptopurines, thioinosine (mercaptopurine riboside), methotrexate, enositabine, cytarabine, ancitabine hydrochloride (cyclocytidine hydrochloride), fluoruracil, 5-FU, tegafur, doxyfluridine, carmofur, etc.
  • Plant-derived anticancer agents such as alkaloids such as vinplastin sulfate, pinklistin sulfate, vindesine sulfate, paclitaxel, taxol, irinotecan hydrochloride, nogitecan hydrochloride, actinomycin D, Mitomycin C, chromomycin A3, bleomycin hydrochloride, bleomycin sulfate, pepromycin sulfate, daunorubicin hydrochloride, doxorubicin hydrochloride, aclarubicin hydrochloride (acracinomycin A), pirarubicin hydrochloride, epilubicin hydrochloride, ne Anticancer antibiotics such as ocarchinostatin, mitoxantrone hydrochloride, carboplatin, cisplatin, L-parasine, Laseparaton, procarbazine hydrochloride, tamoxifen citrate, ube-metas
  • the ribosome of the present invention can be used for the treatment of diseases such as cancer and inflammation.
  • cancer includes all neoplastic diseases such as tumor and leukemia.
  • these drugs are included in the sugar chain-modified ribosome of the present invention and administered, the drugs accumulate at cancer and inflammation sites compared to when the drug is administered alone. Compared to the case of single administration, it can accumulate 2 times or more, preferably 5 times or more, more preferably 10 times or more, and particularly preferably 50 times or more.
  • ribosome (reference ribosome) to which tris (hydroxymethyl) aminomethane is bound, it can accumulate 3 to 4 times, preferably 4 to 6 times.
  • the sugar chain-modified ribosome of the present invention can be used for treatment of various diseases by encapsulating a drug as described above.
  • the drug-encapsulated sugar chain-modified ribosome can be administered by intravenous injection or oral administration. Even when the sugar chain-modified ribosome of the present invention is delivered to an organ when orally administered, the medium transferred into the blood by oral administration shows a tendency similar to that of intravenous injection.
  • the compound having a medicinal effect may be encapsulated in a ribosome or bound to the surface of a liposome.
  • a protein can be bound to the surface in the same manner as the above-mentioned linker binding method, and other compounds can be bound by a known method by using a functional group of the compound. it can.
  • Encapsulation inside the ribosome is performed by the following method. In order to encapsulate drugs, etc.
  • the ribosome preparation obtained by encapsulating a drug or gene that can be used for treatment or diagnosis in the ribosome of the present invention selectively controls the migration to cancer tissues, inflammatory tissues, and various tissues. It is intended to increase the efficacy by concentrating therapeutic drugs or diagnostic agents on target cells and tissues, or to reduce side effects by reducing the uptake of drugs to other cells and tissues. .
  • a label compound such as a fluorescent dye or radioactive compound is bound to the ribosome.
  • the labeled compound-binding ribosome binds to the affected area, the labeled compound is taken into the affected cell, and the disease can be detected and diagnosed using the presence of the labeled compound as an indicator.
  • the present invention can also be used in the health 'food field.
  • the points to be noted when used as an oral medicine should be considered as necessary.
  • a functional food such as a specific health food or a “health food”
  • a functional food, nutritional supplement, or health supplement that is encapsulated or bound to the sugar chain-modified ribosome of the present invention can be used as a food composition.
  • Functional foods, nutritional supplements, or health supplements that can be used in the present invention are limited to those that have been designed to effectively express food functions and are processed and converted. It is.
  • nutraceutical or It can be illustrated as a health supplement. These may be included in the ribosome as they are, or processed products such as extracts may be included. Food compositions containing ribosomes are taken orally.
  • the ribosome used may not be bound to a sugar chain, or may be bound to a sugar chain that enhances intestinal absorption or a sugar chain targeted to a specific tissue or organ.
  • the ribosome of the present invention When the ribosome of the present invention is administered as a food composition, it may be processed into foods such as liquid beverages, gel foods, and solid foods. Moreover, you may process into a tablet, a granule, etc.
  • the food composition of the present invention can be used as a functional food, a nutritional supplement or a health supplement depending on the type of food contained in the ribosome.
  • a ribosome containing DHA can be used as a functional food, nutritional supplement, or health supplement effective for mild senile dementia and memory improvement.
  • the improved cholate dialysis method was used according to a previously reported method (Yamazaki, N., Kodama, M. and Gabius, H. —J. (1994) Methods Enzymol. 242, 56—65).
  • dipalmitoyl phosphatidylcholine, cholesterol, dicetyl phosphate, gandarioside and dipalmitoyl phosphatidylethanolamine were mixed in a molar ratio of 35: 40: 5: 15: 5 to give a total lipid content of 45.6 mg.
  • 46.9 mg of sodium cholate was added, and dissolved in 3 ml 1 of black mouth form Z methanol (1: 1) solution.
  • lipid membrane was resuspended in 3 ml of N tris (hydroxymethyl) 3-aminopropanesulfonic acid buffer (pH 8.4) and stirred at 37 ° C for 1 hour. The solution was then purged with nitrogen and sonicated to obtain a clear micelle suspension. Sarakoko, micelle suspension was ultrafiltered using PM10 membrane (Amicon Co., USA) and ⁇ Tris (hydroxymethyl) 3 aminopropane sulfonate buffer (pH 8.4) (fractionated molecular weight: 10 , 000) to prepare 10 ml of uniform ribosome (average particle size lOOnm).
  • this ribosome solution was subjected to ultrafiltration (fractionated molecular weight: 300,000) with an XM300 membrane and a carbonate buffer (pH 8.5).
  • 40 mg of tris (hydroxymethyl) aminomethane dissolved in 1 ml of carbonate buffer (pH 8.5) was added to 10 ml of ribosome solution.
  • the solution was then stirred at room temperature for 2 hours, then stirred overnight under refrigeration, ultrafiltered with a molecular weight cut off of 300,000 to remove free tris (hydroxymethyl) aminomethane, and the carbonate buffer solution.
  • Example 5 Binding of sugar chains onto ribosome membrane surface-bound human serum albumin (HSA) 2 mg of each sugar chain prepared in Example 4 was dissolved in purified water, and 0.25 g NH HCO was dissolved.
  • HSA human serum albumin
  • a 10 ml portion of the ribosome solution obtained in Example 3 was added to the cross-linking reagent 3, 3, 1 dithiobis (sulfosuccinimidyl propionate (DTS SP; Pierce Co.
  • DTS SP 1, 1 dithiobis (sulfosuccinimidyl propionate
  • Example 6 Hydrophilization treatment on ribosome membrane surface-bound human serum albumin (HSA)
  • HSA human serum albumin
  • the solution (2 mgZml) and (2.5 ml) were mixed and stirred at 37 ° C for 3 hours.
  • This mixed solution was ultrafiltered with a fractional molecular weight 10,000, N-tris (hydroxymethyl) 3 -aminopropanesulfonic acid buffer ( pH 8.4) solution to remove free cy5.5, and cy5.
  • a 5-labeled human serum albumin solution was prepared.
  • Ribosomes have been reported (Yamazaki, N., Kodama, M. and Gabius, H. —J.
  • the obtained lipid membrane was resuspended in 3 ml of TAPS buffered physiological saline (pH 8.4) and stirred at 37 ° C for 1 hour. The solution was then purged with nitrogen and sonicated to give 3 ml of a clear micelle suspension. To this sonicated micelle suspension, cy5.5-labeled HSA solution completely dissolved to 0.2 mgZlml with HSA buffer (pH 8.4) was slowly added dropwise with stirring and mixed uniformly.
  • Fluorescent dye-containing micelle suspension was subjected to ultrafiltration using PM10 membrane (Amicon Co., USA) and TAPS buffered saline ( ⁇ 8.4) (fractional molecular weight: 10 000) to obtain uniform fluorescence 10 ml of a ribosome particle suspension containing the dye was prepared.
  • the particle size and zeta potential of the ribosome particles encapsulating the fluorescent dye in the obtained physiological saline suspension (37 ° C) are converted into zeta potential ⁇ particle size ⁇ molecular weight measuring device (Model Nano ZS, Malvern Instruments Ltd. ,, UK), the particle size was about 65 nm to about 125 nm, and the zeta potential was 40 to 70 mV.
  • Liposomes containing the fluorescent dye prepared in Example 7 1 Oml was ultrafiltered using XM 300 membrane (Amic on Co., USA) and carbonate buffer (pH 8.5) (fractionated molecular weight: 300, The pH of the solution was 8.5. Next, 10 mg of a cross-linking reagent bis (sulfosuccinimidyl) sverley HBS 3 ; Pierce Co., USA) was added and stirred at room temperature for 2 hours. Then, to complete the further chemical binding reaction between 7 ° C De ⁇ lipid on the ribosome film dipalmitoylphosphatidyl E Tano Ruamin and BS 3.
  • this ribosome solution was subjected to ultrafiltration (fractionated molecular weight: 300,000) with an XM300 membrane and a carbonate buffer (pH 8.5).
  • 40 mg of tris (hydroxymethyl) aminomethane dissolved in 1 ml of carbonate buffer (pH 8.5) was added to 10 ml of liposome solution.
  • this solution was stirred at room temperature for 2 hours, then stirred under refrigeration, ultrafiltered with a molecular weight cut off of 300,000 to remove free tris (hydroxymethyl) aminomethane, and the carbonate buffer solution.
  • HSA human serum albumin
  • Free periodate sodium periodate was removed by ultrafiltration (fraction molecular weight: 300, 000) with XM300 membrane and PBS buffer (pH 8.0), and N-tris (hydroxymethyl) -3-a
  • the minopropane sulfonate buffer was replaced with PBS buffer (pH 8.0) to obtain 10 ml of oxidized ribosome.
  • PBS buffer pH 8.0
  • 20 mg of human serum albumin (HSA) / PBS buffer (pH 8.0) was added and reacted at room temperature for 2 hours, and then 2 M NaBH CNZPBS buffer (pH 8.0) 100 1 2 hours at room temperature
  • a sugar chain was prepared by the same procedure as in Example 4.
  • the particle size and zeta potential of the ribosome particles encapsulating the fluorescent dye in the obtained physiological saline suspension (37 ° C) are converted into zeta potential ⁇ particle size ⁇ molecular weight measuring device (Model Nano ZS, Malvern Instruments Ltd. ,, UK), the particle size was about 65 nm to about 125 nm, and the zeta potential was 40 to 70 mV.
  • Example 12 Hydrophilization treatment on ribosome membrane surface-bound human serum albumin (HSA)
  • HSA human serum albumin
  • Tumor (EAT) cells were transplanted 5 ⁇ 10 6 cells and used for experiments 7-10 days later.
  • Nembutal solution was administered 300 1 into the peritoneal cavity of cancer-bearing mice and anesthetized.
  • Pre-dose image data was taken with a fluorescence imaging device eXplore Optix (GE Healthcare). From the tail vein, cy5.5-encapsulated sugar chain-modified ribosome (K1) (2001: equivalent to 750 ⁇ g of lipid) was administered, and image data immediately after administration were collected.
  • K1 cy5.5-encapsulated sugar chain-modified ribosome (K1) (2001: equivalent to 750 ⁇ g of lipid) was administered, and image data immediately after administration were collected.
  • the sugar chain-modified ribosome ( ⁇ 3) showed a weak signal at the tumor site, even without the ability to accumulate at the tumor site. In addition, it gradually accumulated at the tumor site until 1 day later, but it changed without any change between 2 days and 3 days later. This result shows that K1-3 ribosome, not ⁇ 3-3 ribosome, is optimal for imaging the tumor site, and that the sugar chain is specific.
  • mice (BalbZc, female, 8 weeks old) were administered with 200 l (2 mg) of monoclonal antibody (Chondrex) for inducing arthritis in the tail vein.
  • monoclonal antibody Chondrex
  • LPS Lipopolysaccharide 100 1 (50 1) was intraperitoneally administered to mice. Mice developed arthritis 3-4 days after administration.
  • Anesthesia was performed by administering 1/10 Nembutal solution into the peritoneal cavity of arthritic mice.
  • Pre-dose image data was taken with a fluorescence imaging device eXplore Optix (GE Healthcare). From the tail vein, cy5.5-encapsulated sugar chain-modified ribosome (Kl, K2) (501: lipid amount 190 g) was administered, and image data immediately after administration were taken. As a control, The same amount of ribosome to which no sugar chain was bound was administered. The image data was collected over time. All image data were taken from the back foot from the back side.
  • sugar chain-modified ribosomes (K1) which have a low sugar chain-modified bond density, accumulate specifically at the inflammatory site (Fig. 10).
  • K1-3 from another lot sample also accumulated at the site of inflammation. This result showed imaging of the site of inflammation by K1-3 ribosome and reproducibility of the specificity (Fig. 11).
  • K1-2 was accumulated at the site of inflammation even if the sugar chain density was low. From these results, the sugar chain-modified ribosome (K1) has a higher sugar chain modification density, which is lower than the ribosome, and the ribosome is more suitable for imaging of inflammatory sites! (Fig. 12)
  • mice (Confirmation of pharmacokinetics of cy5.5 encapsulated sugar chain-modified ribosome in normal mice) As normal mice, BalbZc, female, 7-8 weeks old were used.
  • Nembutal solution was administered 100 1 into the abdominal cavity of normal mice and anesthetized.
  • Pre-dose image data was taken with a fluorescence imaging device eXplore Optix (GE Healthcare). From the tail vein, cy5.5-encapsulated sugar chain-modified ribosome (2001: lipid amount 750 g) was administered, and image data immediately after administration were taken. The image data was collected over time. All image data were taken from the ventral side.
  • FIG. 14 shows brain data. One hour after administration, it was confirmed that K1 3 and K2-3 were accumulated in the brain. One day after administration, K1-3 was clearly decreased with K2-3, which has a considerable amount of brain remaining. From this result, it was found that sugar chain-modified ribosome (K1) penetrates the blood-brain barrier (BBB) and penetrates into tissues. In addition, the sugar-modified ribosome (K2) decreased after one day, indicating that it cannot pass through the BBB force that enters the brain blood vessels and does not penetrate into the tissue. This proved that there was a specific dynamic in the brain depending on the type of sugar chain. [0244] FIG. 15 shows liver data. One day later, K2-6 showed a strong signal. From this result, it was proved that the specificity of accumulation in the liver due to the difference in sugar chain type and modified bond density can be seen as imaging.
  • BBB blood-brain barrier
  • FIG. 16 shows kidney data. Compared with the K1 and K3 sugar chains, the K2 sugar chain (K2-3) showed a strong signal 1 hour after administration. From this result, it was proved that K2-3 sugar chain can image the specificity of accumulation in the kidney.
  • FIG. 17 shows spleen data. K1-3, 4, 6 and K2-4, 6 were accumulated. This result showed that the specificity of accumulation in the spleen due to the difference in the type of sugar chain and the modified binding density can be seen as an image.
  • Figure 18 shows lung data. For K1-3, the signal persisted 1 hour and 1 day after administration. From this result, it was proved that the specificity of accumulation in the lung due to the difference in the type of sugar chain and the modified bond density can be seen as imaging.
  • FIG. 19 shows spleen data.
  • the signal is weak overall, but partially (K2—3, ⁇ 2
  • FIG. 20 shows cardiac data.
  • the signal without sugar chain showed a stronger signal. From this result, it was found that the specificity of accumulation in the heart due to the difference in the type of sugar chain and the modified binding density can be seen as imaging.
  • FIG. 21 shows K1-3 whole body scan data (other than the parietal region) immediately after administration.
  • K13 has a weaker liver signal than no sugar chain. These results indicate that immediately after administration, K1-3 liposomes are less likely to be taken into the liver than ribosomes without sugar chains.
  • FIG. 22 shows whole body scan data of K1-4, 6 (other than the parietal region) immediately after administration. Like K13, liver signal is weaker than without sugar chain. These results indicate that immediately after administration, sugar chain-modified liposomes (K1) are less likely to be taken up by the liver than ribosomes without sugar chains.
  • FIG. 23 shows whole body scan data of ⁇ 2-3 (other than the parietal region) immediately after administration. No sugar chain The liver signal is stronger than. From these results, it was proved that K2-3 ribosomes were more easily taken into the liver than ribosomes without sugar chains immediately after administration. This result was the same as that shown in FIG. Immediately after administration, specificity to the liver was shown by the type of sugar chain.
  • Fig. 24 shows whole body scan data for K2–4, 6 (except for the parietal region) immediately after administration. K2-4 and K2-6 have weaker liver signals than without sugar chains. These results indicate that immediately after administration, K2-4 and K2-6 ribosomes are less likely to be taken into the liver than ribosomes without sugar chains. Immediately after administration, specificity for the liver due to the difference in the modified binding density was shown.
  • FIG. 25 shows whole body scan data of K3-3 (except for the parietal region) immediately after administration.
  • the signal in the liver is the same as in the case of no sugar chain. From these results, it was found that K3-3 ribosomes were less likely to be taken into the liver immediately after administration compared to ribosomes without sugar chains.
  • FIG. 26 shows whole body scan data for K3-4 and 6 (other than the parietal region) immediately after administration. K3-4 and K3-6 have weaker liver signals than without sugar chains. From these results, it was found that immediately after administration, sugar chain-modified ribosome (K3) is less likely to be taken into the liver than ribosomes without sugar chain.
  • FIG. 27 shows whole body scan data without sugar chain (other than the parietal region) and changes with time. One day later, signals were detected in the liver and bladder. From this result, it was found that ribosomes can be observed by force imaging of metabolism and excretion into the liver and bladder.
  • FIG. 28 shows K13 whole body scan data (other than the parietal region) and time course. One day later, a signal was detected in the bladder. From this result, it was proved that ribosome can be observed by force imaging of metabolism and excretion into the liver and bladder.
  • FIG. 29 shows K 14 whole body scan data (other than the parietal region) and time course. One day later, signals were detected in the liver and bladder. Two days later, the signal in the liver decreased.
  • Figure 30 shows the time course of K1-6 whole body scan data (except for the parietal region). From this result, it was proved that ribosome can be observed by force imaging of metabolism and excretion into the liver and bladder.
  • Micro BCA TM Protein Assay Reagent kit (catalog number 23235BN) (PIERCE Co. LTD) was used for the measurement of protein amount.
  • As a standard substance 2 mg Zml albumin (BSA) attached to the kit was used.
  • a standard substance (2 mgZml: albumin) was diluted with PBS buffer solution to prepare 0, 0.25, 0.5, 1, 2, 3, 4, 5 g / 50 1 solutions.
  • Cy5.5-encapsulated sugar chain-modified ribosomes were diluted 20-fold with PBS buffer to prepare a sample solution.
  • the standard solution and the sample solution were each dispensed into a test tube for 50 minutes.
  • 100 1 of 3% sodium lauryl sulfate solution (SDS solution) was added to each test tube.
  • the test tube was allowed to stand at 60 ° C for 1 hour. After returning to room temperature, the absorbance was measured at 540 nm, a calibration curve was prepared with a standard solution, and the amount of ribosomal protein was measured. The following table shows the results.
  • the amount of ribosome component lipid was calculated by quantifying the amount of cholesterol.
  • Detamina TC555 kit Cat. No. UCCZEAN128, (KYOWA Co. LTD) was used for lipid quantification. 50mgZml attached to kit as standard
  • Lipid content ⁇ 5 ⁇ / ⁇ 1) Cholesterol content gZSO / zl) X 4.51 (Conversion factor) The quantitative results of lipid content are shown below.
  • Ribosome particles were diluted 50-fold with purified water and measured using a Zetasizer Nano (Nan-ZS: MA LVERN Co. LTD).
  • Human serum albumin ZN Tris (hydroxymethyl) 3-aminopropanesulfonic acid buffer (pH8.4) solution (10mgZml), (2ml) with cy5.5ZN-tris (hydroxymethyl) 3 aminopropanesulfonic acid buffer ( pH 8.4) solution (2mgZml) and (2.5ml) were mixed and stirred at 37 ° C for 3 hours.
  • This mixed solution is ultrafiltered with a molecular weight cut-off of 10,000 to remove free cy5.5 and prepare a cy5.5-labeled human serum albumin solution.
  • a sugar chain is prepared in the same manner as in Example 4.
  • Conjugate glycosylated amine compounds to DTSSP on the screen Free sugar chains and tris (hydroxymethyl) aminomethane are removed by ultrafiltration (fraction molecular weight: 300,000) with XM300 membrane and HEPES buffer (pH 7.2). As a result, a ribosome in which a sugar chain, cy5.5-labeled human serum albumin and ribosome are bound is obtained.
  • a fluorescent dye cy3 or cy5 is added to the S—S group in the HSA on the ribosome surface obtained in Example 6 for reaction and labeling.
  • CyX 5.5-encapsulated glycoside-modified ribosome (K1) was prepared using SLX as the sugar chain (see Fig. 40) o
  • the ribosome was isolated from Yamazaki, N. J. Membrance.
  • TAPS tris (hydroxymethyl) methylaminobutane sulfonic acid buffer
  • Cy5.5 was conjugated to HSA using the following labeling method. 20 mg of HSA and 2 mg of CY5.5-NHS ester (GE Healthcare CO., LTD) were dissolved in 3 ml of TAPS (pH 8.4.4) and stirred at 37 ° C for 3 hours. This solution was ultrafiltered with TAPS ( ⁇ 8.4) using an ultrafiltration cell (Model 8010; Amicon CO., LTD) fitted with an Amicon Diaflo PM 10 membrane (Amicon CO., LTD) and remained. Cy5.5.—NHS ester was removed.
  • DTSSP 3,3 dithiobis (sulfosuccinimidylpropionate)
  • PIER CE 3,3 dithiobis (sulfosuccinimidylpropionate)
  • DTSSP was used as a cross-linking agent. 10 mg of DTSSP was added to 10 ml of the ribosome solution, stirred at 20-25 ° C. for 2 hours, and further stirred at 4 ° C. overnight. This solution was ultrafiltered through XM300 (Amicon CO., LTD) to remove the remaining DTSSP. Amination of the reducing group terminal of the sugar chain was performed by glycosylamination reaction. 2 mg of SLX (Calbiochem CO., LTD) was dissolved in 0.5 mL of distilled water. Add 25 g NH HCO and 37

Abstract

L'invention concerne un produit pour l'imagerie moléculaire. Plus précisément, l'invention concerne un liposome modifié par une chaîne de sucre comprenant un liposome, un groupe chaîne de sucre, un groupe protéine de liaison et un groupe composé hydrophile, le groupe protéine de liaison se fixant à la face externe du liposome, le groupe chaîne de sucre se fixant à au moins une partie du groupe protéine de liaison et le groupe composé hydrophile se fixant à la face externe du liposome ou à une partie du groupe protéine de liaison. Dans un autre aspect, l'invention concerne une composition médicinale qui comprend le liposome modifié par une chaîne de sucre ainsi qu'un ingrédient actif du point de vue pharmaceutique. Dans encore un autre aspect, l'invention concerne un liposome modifié par une chaîne de sucre à utiliser en tant que produit pour l'imagerie moléculaire. Dans encore un autre aspect, l'invention concerne un procédé de production d'un liposome contenant une chaîne de sucre marquée.
PCT/JP2007/052289 2006-02-08 2007-02-08 Liposome modifié par une chaîne de sucre convenant pour l'imagerie moléculaire et utilisation et production de celui-ci WO2007091661A1 (fr)

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WO2009022756A1 (fr) * 2007-08-13 2009-02-19 Katayama Chemical Industries Co., Ltd. Diagnostic et traitement d'un trouble ischémique
WO2009025168A1 (fr) * 2007-08-20 2009-02-26 Konica Minolta Holdings, Inc. Liposome modifié par un sucre contenant un composé d'agent de contraste et un agent de contraste
WO2009104649A1 (fr) * 2008-02-22 2009-08-27 片山化学工業株式会社 Liposomes synthétiques contenant des glycolipides
WO2009133867A1 (fr) * 2008-05-01 2009-11-05 片山化学工業株式会社 Liposome contenant un colloïde de métal
WO2009148169A1 (fr) * 2008-06-06 2009-12-10 片山化学工業株式会社 Technique de traitement de tumeurs utilisant un complexe ammine-platine à haute concentration encapsulé dans un liposome
EP2228074A1 (fr) * 2009-02-19 2010-09-15 Korea Institute of Science and Technology Conjugué de protéines ciblant la tumeur et son procédé de préparation
JP2010215572A (ja) * 2009-03-17 2010-09-30 Japan Health Science Foundation 歯髄炎診断マーカー及び歯髄炎診断システム
WO2011128931A1 (fr) * 2010-04-12 2011-10-20 財団法人ヒューマンサイエンス振興財団 Marqueur diagnostique de pulpite et système diagnostique de pulpite
WO2011152046A1 (fr) * 2010-05-31 2011-12-08 国立大学法人千葉大学 Sonde fluorescente pour imagerie de ganglions lymphatiques
WO2012066896A1 (fr) 2010-11-19 2012-05-24 Kato Junji Préparation pharmaceutique combinée

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