WO2007091661A1

WO2007091661A1 - Sugar chain-modified liposome suitable for molecular imaging and utilization and production of the same

Info

Publication number: WO2007091661A1
Application number: PCT/JP2007/052289
Authority: WO
Inventors: Noboru Yamazaki; Kazunori Oie
Original assignee: National Institute Of Advanced Industrial Science And Technology; Katayama Chemical Industries Co., Ltd.
Priority date: 2006-02-08
Filing date: 2007-02-08
Publication date: 2007-08-16
Also published as: JPWO2007091661A1

Abstract

It is intended to provide a molecular imaging agent. Namely, there is provided a sugar chain-modified liposome comprising a liposome, a sugar chain group, a linker protein group and a hydrophilic compound group, wherein the linker protein group attaches to the outer face of the liposome, the sugar chain group attaches to at least a part of the linker protein group, and the hydrophilic compound group attaches to the outer face of the liposome or a part of the linker protein group. In another aspect, a medicinal composition which comprises the sugar chain-modified liposome together with a pharmaceutically active ingredient is provided. In a still another aspect, a sugar chain-modified liposome to be used as a molecular imaging agent is provided. In a still another aspect, a method of producing a liposome containing a labeled sugar chain is provided.

Description

Specification

Glycosylated ribosomes suitable for molecular imaging and their use and production

Technical field

[0001] The present invention relates to a ribosome. Specifically, 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.

Background art

[0002] As an example of a specific goal to be realized by the US National Nanotechnology Strategy (NNI), “Drug and gene delivery system (DDS: drug delivery system) targeting and targeting cancer cells and target tissues” . 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”. On the other hand, 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. These materials are also expected to be used for diagnosis as well as treatment.

[0003] The therapeutic effect of a drug is expressed by the drug reaching a specific target site and acting there. On the other hand, the side effect of drugs is that the drug acts on unnecessary parts. Therefore, the development of a drug delivery system is required for effective and safe use of drugs. In particular, targeting DD (targeting) DD S is the concept of V when drug is delivered "to the necessary site in the body", "necessary amount", "only for the required time". Liposome, which is a fine particle carrier as a representative material for that purpose, has attracted attention. In order to give this particle a targeting function, Passive targeting methods such as changing the quality type, composition ratio, particle size, and surface charge have been tried, but this method is still insufficient and further improvement is required.

[0004] On the other hand, active targeting methods have also been tried to enable highly functional targeting! This is called an “missile drug” and is an ideal targeting method, but it has not been completed in Japan and overseas, and future development is expected greatly. In this method, ligands are bound on the surface of the ribosome membrane and are specifically recognized by the receptors present on the cell membrane surface of the target tissue, thereby enabling targeting actively. As a ligand of a receptor present on the cell membrane surface as a target in this active targeting method, an antigen, an antibody, a peptide, a glycolipid, a glycoprotein, or the like can be considered. Among these, 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.

[0005] In addition, since research on various lectins (sugar chain recognition proteins) such as selectin, siglec, and galectin as receptors existing on the cell membrane surface of each target tissue has progressed, The sugar chain having the molecular structure has attracted attention as a new U and DDS ligand (see Non-Patent Document 1 and Non-Patent Document 2).

[0006] With regard to ribosomes having a ligand bound to the outer membrane surface, much research has been conducted as a DDS material for selectively delivering drugs, genes, and the like to target sites such as cancer. However, most of them bind to target cells in vitro but are not targeted to target cells or tissues expected in vivo (see Non-Patent Document 3). In the research and development of DDS materials that utilize the molecular recognition function of sugar chains, some studies have been made on ribosomes into which glycolipids with sugar chains have been introduced. In vitro), and research on ribosomes that have introduced glycoproteins with sugar chains is almost advanced, see Nada (Non-patent document 4, Non-patent document 5, Non-patent document 6 and Non-patent document 7). ) Therefore, systematic research including preparation methods and in vivo analysis of ribosomes bound to a wide variety of sugar chains of glycolipids and glycoproteins has not been developed so far, and future progress has been made. Expected important It is a problem. Furthermore, as a new type of DDS material research, the development of DDS materials that can be used by oral administration, which is the simplest and cheapest to administer, is also an important issue. For example, 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).

[0007] 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. However, in this pharmaceutical composition, 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.

[0008] 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.

[0009] Therefore, there is a demand for efficiently designing and providing sugar chain-modified ribosomes that are optimal for molecular imaging.

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, Ν. V., Andre, S., Gabius, S. and Gabius, H. —J. (2000) Adv. Drug Delivery Rev. 43 , 225— 24 Non-Patent Document 2: Yamazaki, N., Jigami, Υ., Gabius, Η. —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

Nagy, J. O. (1996) J. Med. Chem. 39, 1018—1020.

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

Disclosure of the invention

Problems to be solved by the invention

Accordingly, 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. Means for Solving the Problems

[0011] In order to solve the above-mentioned problems, 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. [0012] 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.

In order to achieve the above object, the present invention provides, for example, the following means.

[0014] (Item 1)

A sugar chain-modified ribosome, the sugar chain-modified ribosome,

Ribosome and

Sugar chain and

Linker with protein group

A hydrophilic compound group,

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, and the linker protein group binds to the outer surface of the ribosome or part of the linker protein group. A sugar chain-modified ribosome to which a hydrophilic compound group is bound.

(Item 2)

The sugar chain-modified ribosome includes structure I and structure Π,

The structure I is

X-CH NH— R ¹ — NH— C (= 0) — R ² — C (= 0) — NH— R ³

2

X is capable of CH—NH binding with the linker protein contained in the ribosome.

2

A group in which the functional group a is removed from the structural unit containing the functional group a,

R ¹ is the linker protein group,

R ² is a linker-protein cross-linking group,

R ³ is the sugar chain group; and

The structure II is

Y-NH-C (= 0)-R ⁴ -C (= 0)-NH-R ⁵

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匕合product groups, glycosylation ribosome of claim 1.

(Item 3) Item 2. The sugar chain-modified ribosome according to Item 1, wherein the ribosome has fluorescence.

(Item 4)

Item 4. The sugar chain-modified ribosome according to Item 3, wherein the fluorescence is imparted by a fluorescent dye compatible with the ribosome.

(Item 5)

The fluorescent dye is

[Chemical 10-1] Cy5. 5>

[Chemical 10-2]

<cy3>

cy5, cy ,, cydB, cy3.5, Alexa Fmor350, Alexa Fluor488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor555, Alexa Fluor568, Alexa Fluor 5 94, Alexa Fluor633, Alexa Fluor647, Alexa Fluor680, Alexa Fluor70 Item 5. 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.

(Item 6)

The fluorescent dye is

[Chem 10-3] Ku5. 5>

The sugar chain-modified ribosome according to item 5, wherein

(Item 7)

The sugar chain-modified ribosome according to Item 4, wherein the fluorescent dye is encapsulated in ribosome (Item 8)

Wherein R ¹ is a mammalian derived protein groups, glycosylation ribosome of claim 2. (Item 9)

Wherein R ¹ is a human-derived protein groups, glycosylation ribosome of claim 8.

(Item 10)

Wherein R ¹ is a human-derived serum protein groups, glycosylation ribosome of claim 9. (Item 11)

Wherein R ¹ is a sugar chain modification ribosome of claim 8, which is a serum albumin group.

(Item 12)

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)

Item 13. The sugar chain-modified ribosome according to Item 12, wherein R ² is a 3, 3, -dithiobis (sulfosuccinimidyl propionate) group.

(Item 14)

Wherein 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.

(Item 15)

Wherein 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.

(Item 16)

Item 14 wherein 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. The sugar chain-modified ribosome described in 1.

(Item 17)

Item 14 is that wherein R ³ is an α 1-6 mannobiose group, and the α 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. The sugar chain-modified ribosome described.

(Item 18)

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.

(Item 19)

Wherein R ⁴ is bis (sulfosuccinimidyl I succinimidyl) scan base rate group, glycosylation ribosome of claim 18.

(Item 20)

Item 3. The sugar chain-modified ribosome according to Item 2, wherein R ⁵ is a tris (hydroxyalkyl) alkylamino group.

(Item 21)

Tris (hydroxyalkyl) alkylamino group Hydroxyalkyl is C to C hydride

1 6 Roxyalkyl, where alkylamino is a C to C alkylamino.

1 6

The sugar chain-modified ribosome listed.

(Item 22) The sugar chain-modified ribosome according to Item 21, which is a no-group.

(Item 23)

The sugar chain modification according to Item 2, wherein the functional group a has one —CH 2 —C (= 0) —CH group.

twenty two

Ribosome.

(Item 24)

Item 24. The sugar chain-modified ribosome according to Item 23, wherein X is gandioside.

(Item 25)

Item 3. The sugar chain-modified ribosome according to Item 2, wherein the functional group b is an amino group.

(Item 26)

Item 26. The sugar chain-modified liposome according to Item 25, wherein Y is phosphatidylethanolamine.

(Item 27)

2. The sugar chain-modified ribosome according to item 1, comprising the ribosome force dipalmitoyl phosphatidylcholine, cholesterol, ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine and sodium cholate. (Item 28)

28. 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.

(Item 29)

R ² is a 3, 3, 1 dithiobis (sulfosuccinimidyl propionate) group,

(Item 30)

R ³ is selected from the group consisting of a sialyl Lewis X group, a Ν-acetyl lactosamine group, an α 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.

(Item 31)

R ² is a 3,3,1 dithiobis (sulfosuccinimidylpropionate) group, and R ³ is a sialyl Lewis X group, a ァ -acetyllactosamine group, an α 1-6 mannobiose group, and Selected from the group of their combination power, and

(Item 32)

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, and R ³ is a sialyl Lewis X group, a ァ -acetyllactosamine group, α 1-6 mannobiose Selected from the group consisting of groups and their combination powers, and

(Item 33)

R ¹ is a serum albumin group,

R ² is a 3,3,1 dithiobis (sulfosuccinimidylpropionate) group, and R ³ is a sialyl Lewis X group, an N-acetyllactosamine group, an α 1-6 mannobiose group, and Selected from the group consisting of those combinations,

R ⁴ is a bis (sulfosuccinimidyl) suberate group, and

Wherein R ⁵ is a tris (hydroxymethyl) Aminometan group, glycosylation liposome of claim 2.

(Item 34)

The sugar chain-modified ribosome is

[Chemical 10-4]

<Cy5. 5>

34. The sugar chain-modified ribosome according to item 33, which is labeled with

(Item 35)

In the ribosome, the ratio of protein to lipid is about 0.1 to about 0.5. The sugar chain-modified ribosome according to Item 1.

(Item 36)

2. The sugar chain-modified ribosome force according to item 1, wherein the sugar chain-modified ribosome has a particle diameter of about 80 nm to about 165 nm in the maximum range of the particle size distribution of the ribosome.

(Item 37)

The sugar chain-modified ribosome according to Item 1, wherein the sugar chain-modified ribosome has an average particle size of about 50 nm to about 300 nm.

(Item 38)

Item 38. An imaging agent comprising the sugar chain-modified ribosome according to any one of items 1 to 37. (Item 39)

40. A 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.

(Item 40)

40. The composition of item 39, wherein the desired substance is a diagnostic agent or a research reagent. (Item 41)

41. The 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.

(Item 42)

40. The composition of item 39, for use in molecular or in vivo imaging.

(Item 43)

40. The composition of item 39, wherein the substance comprises the biological agent.

(Item 44)

38. 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 45)

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 pharmaceutical composition according to 4.

(Item 46)

38. Use of the sugar chain-modified ribosome according to any one of items 1 to 37 for the production of a composition for labeling a desired site.

(Item 47)

49. Use according to item 46, wherein the desired site is selected from the group consisting of brain, liver, kidney, spleen, lung, spleen, heart, inflammatory site and tumor site force.

(Item 48)

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. And the desired site is selected from the group consisting of brain, liver, kidney, spleen, lung, spleen, heart, inflammatory site and tumor site force.

(Item 49)

A method for producing a sugar chain-modified ribosome, the method comprising:

(a) a step of obtaining a lipid membrane by suspending lipids in a methanol Z chloroform solution and stirring, evaporating the stirred solution, and drying the precipitate in vacuo;

(b) suspending the lipid membrane in a suspension buffer and sonicating;

(c) providing the fluorescently labeled liposome by mixing the sonicated solution and the fluorescently labeled solution;

(d) a step of hydrophilic treatment of the ribosome with tris (hydroxyalkyl) aminoalkane;

(e) binding a linker protein to the hydrophilic ribosome treated to produce a linker protein-binding ribosome; and

(f) A method comprising a step of binding a sugar chain to the ribosome to produce a sugar chain-modified ribosome.

(Item 50)

The fluorescent labeling solution of step (c) is [Chem. 10-5] Ku5. 5>

50. A method for producing a sugar chain-modified ribosome according to item 49, comprising:

(Item 51)

Item 52. The method according to Item 49, wherein the linker protein power in step (e) is human serum albumin.

(Item 52)

A method for producing a sugar chain-modified ribosome for delivering a drug to a target delivery site, the method comprising:

(a) providing fluorescently labeled sugar chain-modified ribosomes having various sugar chain densities to achieve delivery to the target delivery site, comprising:

(i) a step of obtaining a lipid membrane by suspending lipids in methanol Z chloroform solution and stirring, evaporating the stirred solution, and drying the precipitate in vacuum;

(ii) suspending the lipid membrane in a suspension buffer and sonicating;

(iii) a step comprising mixing the sonicated solution and the fluorescent labeling solution

(b) determining the density of the sugar chain on the sugar chain-modified ribosome to achieve optimal delivery to the delivery site; and

(c) a step of producing a drug-containing liposome by incorporating the drug into the determined optimal sugar chain-modified ribosome

Including the method. (Item 53)

A method for producing a fluorescent dye-containing sugar chain-modified ribosome,

A) the step of forming a force that encapsulates the fluorescent dye in the ribosome or a bound ribosome;

B) Hydrophilizing the ribosome;

C) binding the ribosome and linker protein; and

D) Step of binding a sugar chain to the ribosome

Including the method.

(Item 54)

54. A method according to item 53, comprising the step of filter filtration subsequent to the step D).

(Item 55)

Step A)

(A1) Dipalmitoyl phosphatidylcholine, cholesterol, gandarioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine and sodium cholate are mixed at a molar ratio of 35: 40: 15: 5: 5: 167 Suspending in form (1: 1) solution;

(A2) Evaporate the chloroform'methanol solution, vacuum dry, and resuspend in N-tris (hydroxymethyl) 3-aminopropanesulfonate buffer (pH 8.4) to form a resuspension Process;

(A3) The resuspension is stirred at 30 ° C. to 40 ° C., purged with nitrogen, and sonicated; (A4) The fluorescent dye is contained in the solution sonicated in (A3) A step of mixing a fluorescent dye solution, ultrafiltering the mixed solution with a molecular weight cut-off of 10,000, and preparing a ribosome containing the fluorescent dye;

55. A method for producing a fluorescent dye-containing sugar chain-modified ribosome according to Item 53 or 54, comprising

(Item 56)

The fluorescent dye solution in the step (A4) is a fluorescent dye standard labeled with a fluorescent dye. Is a solution containing a protein, the following:

(1) 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

(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,

56. The method of item 55, which is prepared by a process comprising:

(Item 57)

Step B) includes the following:

(B1) The solution containing force or bound ribosome encapsulating the fluorescent dye is ultrafiltered with a fractionation amount of 300,000, and the buffer contained in the solution is carbonate buffer (pH 8.5) Replacing with

(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;

(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 (ρΗ8. 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;

The method for producing a fluorescent dye-containing sugar chain-modified liposome according to any one of items 53 to 56, comprising:

(Item 58) Step B) includes the following:

(Bl ') The solution containing the fluorescent dye or the solution containing the bound ribosome should be separated and dissociated twice under the conditions of a fractional amount of 100,000, 2000 X g and 60 minutes. A step of exchanging the buffer contained in the solution with a carbonate buffer (PH8.5);

(B2 ′) Addition of bis (sulfosuccinimidyl) suberate to the solution in which the buffer solution is converted to the carbonate buffer solution in the step (B1 ′) After stirring at 37 ° C, the mixture was subjected to ultrafiltration by separating twice, with a molecular weight cut off of 100,000, 2000 X g, 60 minutes, by force, and free bis (sulfosuccin Imidyl) suberate removal;

(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;

58. The production method according to items 53 to 57, comprising:

(Item 59)

Step C) includes the following:

(C1) Add a solution of sodium metaperoxide ZN-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) to a solution containing force or bound ribosome containing the fluorescent dye. Adding and acidifying the surface of the ribosome particles by refrigeration to stirring at room temperature overnight;

(C2) In the step (C1), 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);

(C3) In the step (C2), 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

(C4) Further, sodium cyanoboronate ZPBS buffer (pH 8.0) was added to the reaction solution, stirred at refrigeration to room temperature, ultrafiltered with a molecular weight cut off of 300,000, and free Removing sodium boroborate and human serum albumin, and replacing the buffer of the solution with carbonate buffer (PH8.5)

59. The production method according to items 53 to 58, comprising:

(Item 60)

Step C) includes the following:

(C4) Further, the 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

The manufacturing method of item 53-59 including this.

(Item 61)

Step C) includes the following:

(C1 ') Add a solution of sodium metaperiodate ZN-tris (hydroxymethyl) -3-aminopropanesulfonate buffer (pH 8.4) to the solution containing the force or bound ribosome containing the fluorescent dye. And stir overnight at refrigerated to room temperature to oxidize the ribosome particle surface. The step of:

In the (C2 ′) (CI ′) step, 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). ;

(C3 ′) In the step (C2 ′), 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;

(C4) Further, sodium cyanoborate ZPBS buffer (pH 8.0) was added to the reaction solution, and the mixture was separated twice under the conditions of a molecular weight cutoff of 100,000, 2000 × g for 60 minutes f¾, Ultrafiltration by force of separation to remove free sodium cyanoboronate and human serum albumin, and exchange the buffer of the solution with carbonate buffer (pH 8.5)

The manufacturing method of item 53-59 including this.

(Item 62)

Step C) includes the following:

(C1 ') Add a solution of sodium metaperiodate ZN-tris (hydroxymethyl) -3-aminopropanesulfonate buffer (pH 8.4) to the solution containing the force or bound ribosome containing the fluorescent dye. And acidifying the surface of the ribosome particles by stirring overnight at refrigerated to room temperature;

(C2 ′) In the (C1 ′) step, the solution containing the ribosome whose surface is oxidized is subjected to two separations under the conditions of a molecular weight cutoff of 100,000 and 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). ;

(C4 ′) Further, the 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. The production method according to items 53 to 59, comprising a step of removing rubumin and exchanging the buffer solution of the solution with a carbonate buffer solution (pH 8.5).

(Item 63)

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;

(D2) Add 3, 3 'dithiobis (sulfosuccinimidyl propionate) to the solution containing the force or bound ribosome containing the fluorescent dye, and stir at refrigeration to approximately 37 ° C. And ultrafiltering with a molecular weight cut off of 300,000 to remove the free 3,3′-dithiopis (sulfosuccinimidyl propionate); and

(D3) In the step (D2), 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;

(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),

63. The production method according to items 53 to 62, which comprises

(Item 64)

64. The production method according to items 53 to 63, further comprising the step of hydrophilicizing the ribosome to which the sugar chain is bound following the step D).

(Item 65)

A method for producing a fluorescent dye-containing sugar chain-modified ribosome, the production method comprising:

A) forming a ribosome in which a fluorescent dye is encapsulated in the ribosome;

B) Hydrophilizing the ribosome;

C) binding the ribosome to a linker protein;

D) a step of binding a sugar chain to the ribosome;

E) Hydrophilizing the ribosome to which the sugar chain is bound; and F) Filtering the solution containing the hydrophilic ribosome

Manufacturing method.

(Item 66)

Step A) includes the following:

(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. In the step of preparing ribosome, 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. Comprising a step prepared by the step of

Step B) includes the following:

(B1) The solution containing the ribosome encapsulating or binding the fluorescent dye is ultrafiltered at a fractional fraction of 300,000, and the buffer contained in the solution is carbonate buffer (pH 8.5). ) To replace with;

(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 (ρΗ8. 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:

(C1) A solution containing sodium phosphoperiodate ZN tris (hydroxymethyl) 3-aminopropanesulfonate buffer solution (PH8.4) is added to a solution containing force or bound ribosome encapsulating the fluorescent dye, Stir overnight under refrigeration to acidify the ribosome particle surface;

(C3) In the step (C2), 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

(C4) To the reaction solution was added sodium cyanoboronate ZPBS buffer (pH 8.0), stirred at refrigeration to room temperature, ultrafiltered with a molecular weight cut off of 300,000, and free cyanoboronic acid. Removing sodium and the human serum 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;

(D2) 3, 3′-dithiobis (sulfosuccinimidyl propionate) is added to a solution containing a ribosome containing or bound to the fluorescent dye, and refrigerated to about 37 ° C. Stir with And ultrafiltering with a molecular weight cut off of 300,000 to remove the free 3, 3, -dithiopis (sulfosuccinimidyl propionate); and

(D3) In the step (D2), 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

(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.

(Item 67)

Step A) includes the following:

(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. In the step of preparing ribosome, 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. Comprising a step prepared by the step of 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. 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 (ρΗ8. 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:

(C4) Further, the 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:

(D2) 3, 3′-dithiobis (sulfosuccinimidyl propionate) is added to a solution containing a ribosome containing or bound to the fluorescent dye, and refrigerated to about 37 ° C. And removing the free 3, 3, -dithiopis (sulfosuccinimidyl propionate) by ultrafiltration with a molecular weight cut off of 300,000; and

(Item 68)

Step A) includes the following:

(A2) The chloroform'methanol solution is evaporated, dried under reduced pressure, and resuspended in N-tris (hydroxymethyl) 3-aminopropanesulfonate buffer (pH 8.4) to form a resuspension. Process;

(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. In the step of preparing ribosome, 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. Comprising a step prepared by the step of

Step B) includes the following:

(B1 ′) The solution containing the fluorescent dye or the solution containing the bound ribosome is limited by separating and separating the solution twice under conditions of a molecular weight cutoff of 100,000, 2000 × g for 60 minutes. A step of filtering and replacing the buffer contained in the solution with carbonate buffer (pH 8.5);

(B2 ′) Addition of bis (sulfosuccinimidyl) suberate to the solution in which the buffer solution is converted to the carbonate buffer solution in the step (B1 ′) After stirring at 37 ° C, the mixture was subjected to ultrafiltration by detaching it twice under conditions of a molecular weight cut off of 100,000, 2000 xg for 60 minutes, and the free bis (sulfosuccinic acid). A step of removing (midyl) suberate;

(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:

(C2 ′) In the (C1 ′) step, a solution containing ribosomes whose surface is oxidized is separated. Molecular weight 100,000, 2000 x g Twice under conditions of 60 min for 60 minutes, ultrafiltration is carried out to remove the separation, the free sodium metaperiodate is removed, and the N tris (hydroxymethyl) E) Step of exchanging 3-aminopropanesulfonic acid buffer with PBS buffer (pH 8.0); (C3 ′) In step (C2 ′), the buffer solution is replaced with the PBS buffer solution. Adding human serum albumin / PBS buffer (pH 8.0) and reacting at refrigeration to room temperature to form a reaction solution;

(C4) Further, sodium cyanoborate ZPBS buffer (pH 8.0) was added to the reaction solution, and the mixture was separated twice under the conditions of a molecular weight cutoff of 100,000, 2000 × g for 60 minutes f¾, Including ultrafiltration by forcing separation, removing free sodium cyanoborate and human serum albumin, and replacing the buffer of the solution with carbonate buffer (pH 8.5), D) The process is as follows:

(D3) In 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

(Item 69)

Step A) includes the following:

(A1) Dipalmitoyl phosphatidylcholine, cholesterol, gandioside, disetti Ruphosphate, dipalmitoyl phosphatidylethanolamine and sodium cholate are mixed in a molar ratio of 35: 40: 15: 5: 5: 167 and suspended in a methanol 'black form (1: 1) solution. Process;

Step B) includes the following:

(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 to remove free tris (hydroxymethyl) aminomethane, and the carbonate buffer Including the step of replacing N tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) to produce a solution containing ribosomes that have been treated with hydrophilicity, and the step C). But the following:

(C4 ′) Further, the 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:

(D3) In 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

(Item 70)

A) forming ribosomes;

B) Hydrophilizing the ribosome;

C) a step of binding the ribosome and a linker protein, wherein the linker protein is labeled with a fluorescent dye; and

D) Step of binding a sugar chain to the ribosome

Including the method.

(Item 71)

39. The imaging agent according to Item 38, wherein the sugar chain of the sugar chain-modified ribosome is a sialyl Lewis X group, and the sialyl Lewis X group has a modified binding density of 0.025 mg sugar chain Z mg lipid.

(Item 72)

72. The imaging agent according to item 71, for imaging an inflamed site or cancer tissue. (Item 73)

73. The imaging agent according to item 72, wherein the inflammatory site or cancer tissue contains a parenchyma.

(Item 74)

40. The 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.

(Item 75)

75. A composition according to item 74, for delivering the substance to an inflamed site or cancer tissue. (Item 76) 76. A composition according to item 75, wherein the inflammatory site or cancer tissue comprises a parenchyma.

(Item 77)

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.

(Item 78)

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. 80. The carrier according to item 77, for delivering a drug.

(Item 79)

80. A carrier according to item 77, wherein the inflammatory site or cancer tissue comprises a parenchyma.

(Item 80)

80. The carrier according to item 79, wherein the labeling substance is fluorescent.

(Item 81)

A system for molecular or in vivo imaging of a site of interest comprising:

A) a sugar chain-modified ribosome specific to the target site;

B) sign; and

C) means for examining the presence or absence of the label;

Where, where

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.

system.

(Item 82)

82. A system according to item 81, wherein the label is a fluorescent substance.

(Item 83)

83. The system according to Item 82, wherein 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 Z mg lipid. Mu.

(Item 84)

84. A system according to item 81, for imaging an inflamed site or cancer tissue.

(Item 85)

85. The system of item 84, wherein the inflammatory site or cancer tissue comprises parenchyma.

(Item 86)

84. A system according to item 81, wherein the means for checking for the presence of the marker includes a scanning microscope (item 87).

89. A system according to item 86, wherein the means for checking the presence of the sign further comprises a stick objective lens.

(Item 88)

84. The system according to Item 81, wherein the means for examining the presence or absence of the label is a means for detecting fluorescence.

(Item 89)

54. 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

d) A step of binding a sugar chain to the linker protein in the ribosome to produce the fluorescent dye-containing sugar chain-modified ribosome

Including

The production method, wherein the steps b) to c) are performed in an arbitrary order.

(Item 90)

54. 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

e) hydrophilizing the fluorescent dye-containing sugar chain-modified ribosome; and

f) A step of filtering the solution containing the hydrophilic dye-containing sugar chain-modified ribosome containing the fluorescent dye

Including

(Item 91)

Step c) and step b) are performed in order after step a).

The c) step is as follows:

(cl) adding powder A containing carbonate buffer solution to powder A containing 3,3,1 dithiobis (sulfosucci-midylpropionate) to prepare a mixed solution; and

(c2) The mixed solution is added to a solution containing ribosome, stirred at room temperature for 16 to 20 hours, ultrafiltered with a molecular weight cut off of 30,000, desalted, and 3, 3, -dithiopis ( Including a step of preparing a solution containing a fluorescently encapsulated ribosome bound with sulfosuccinimidylpropionate)

The step b) includes the following:

(bl) concentrating the solution containing the fluorescently encapsulated ribosome and adding the solution A to the concentrated solution containing the fluorescently encapsulated liposome;

(b2) The mixed solution containing the fluorescently encapsulated ribosome to which the solution A is added is centrifuged at a fractional molecular weight of 300,000, ultrafiltered and concentrated, and the concentrated mixed solution is mixed with the solution. A step of adding A to prepare the fluorescently encapsulated ribosome that has been made hydrophilic;

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;

(d3) The aminated sugar chain solution is added to the solution containing the hydrophilicized fluorescently encapsulated ribosome, mixed, and then reacted at room temperature for 2 to 6 hours; and a reaction solution step; and

(d4) Solution B containing Tris buffer is added to the reaction solution obtained in the step (d3), and the mixture is stirred at room temperature for 2 to 6 hours, and further stirred for 16 to 48 hours under refrigeration. And a step of ultrafiltration with a molecular weight cut off of 30,000, desalting to produce the fluorescent dye-containing sugar chain-modified ribosome,

Step e)

(el) Concentrate the solution containing the fluorescent dye-containing sugar chain-modified ribosome and add solution C containing 2- [4- (2-hydroxyxetyl) -1-piperajuryl] ethanesulfonic acid (HEPES) buffer Then, ultrafilter with a molecular weight cut off of 30,000, concentrate, add the solution C to the concentrated solution containing the fluorescent dye-containing sugar chain-modified ribosome, and add the fluorescent endothelium to which the sugar chain is bound. Comprising hydrophilizing the type ribosome,

Item 91. The manufacturing method according to Item 90.

(Item 92)

A kit for producing a fluorescent dye-containing sugar chain-modified ribosome,

i) a means for encapsulating or binding the fluorescent dye in the ribosome;

ii) a hydrophilizing agent for the ribosome;

iii) the ribosomal linker protein, and

iv) sugar chains;

V) Means for binding the sugar chain to the ribosome

A kit comprising:

(Item 93)

A kit for producing a fluorescent dye-containing sugar chain-modified ribosome comprising:

A) a solution containing a ribosome bound to a linker protein; B) Powder A containing 3, 3, 1 dithiobis (sulfosuccimid-midylpropionate) and

C) Solution A containing carbonate buffer and

D) solution B containing Tris buffer and

E) A kit comprising 2- [4 (2-hydroxyethyl) 1-piperazyl] ethanesulfonic acid (HEPES) buffer solution C.

The invention's effect

[0020] 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.

Hereinafter, preferred embodiments of the present invention will be described. However, those skilled in the art can appropriately implement the embodiments of the present invention and the well-known technical skills in the field, and the effects and advantages of the present invention can be easily achieved. It should be recognized that you understand.

Hereinafter, embodiments of the present invention will be described in detail.

Brief Description of Drawings

[0021] 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. In the color imaging diagram, the upper red color of the bar is the lower color with the strongest signal. , Indicating that the fluorescent signal is weak. In the black-and-white imaging diagram, the fluorescence signal becomes weaker as the white color above the bar becomes stronger and the lower it becomes. Units

, Photon count, photon / second (ph / s): Counted per second: ¾: number of photon photons).

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. In the color imaging diagram, the red color at the top of the bar is the lower color with the strongest signal, indicating that the fluorescence signal is weaker. In the black-and-white imaging diagram, 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. 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. In 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 Immediately after somal administration, 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 4th stage after administration of ribosomes without sugar chains 2 days, right 5th stage: 3 days after administration of ribosomes without sugar chains. 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. In 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. In the color imaging diagram, the red color above the bar is the lower color with the strongest signal, indicating that the fluorescence signal is weaker. In the black-and-white imaging diagram, 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. In the color imaging diagram, the red color above the bar is the lower color with the strongest signal, indicating that the fluorescence signal is weaker. In the black-and-white imaging diagram, 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. In the black-and-white imaging diagram, 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. In the black-and-white imaging diagram, 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. In the black-and-white imaging diagram, 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. 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 . In each 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, and 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. In the black and white imaging diagram, the whiter the bar, the stronger the fluorescent signal, and 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. In the black-and-white imaging diagram, 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. 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. In the black-and-white imaging diagram, 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. 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. 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. In the black-and-white imaging diagram, 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, K2—3 liposome administration, K2— 4 ribosome administration, K2-6 ribosome administration, unadministered, lower right: K3 ribosome administration 1 day later, left side force is also without glycans, K3-3 ribosome administration, K3 -4 ribosome administration, K3-6 Ribosome administration, untreated. The rightmost bar shows the fluorescence signal intensity obtained by imaging. In the color imaging diagram, 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 ribosome administration, right 2 Stage: 1 hour after administration of K3 ribosome, no sugar chain from left side Ribosome administration, K3-4 ribosome administration, K3-6 ribosome administration, left 3rd stage: K1 liposome administration 1 day later, left side force is also sugar chain Ribosome administration, K1-3 ribosome administration, K1-4 liposome administration, K1-6 ribosome administration, unadministration, middle 3rd stage: K2 ribosome administration 1 day later, left side force is also glycoside-free ribosome administration, K2-3 ribosome Administration, K2—4 ribosome administration, K2—6 ribosome administration, not administered, right third stage: K3 ribosome administration 1 day later, no glycans from the left side, K3—3 ribosome administration, K3—4 ribosome administration, K3— 6 Liposome administration, no administration, left 4th stage: K1 ribosome administration 2 days later, left side force No sugar chain ribosome administration, K1-3 ribosome administration, K1-4 ribosome administration, K1-6 ribosome administration, central 4th stage Eye: 2 days after administration of K2 ribosome, administration of ribosome without sugar chain, administration of K2-3 ribosome, administration of K2-4 ribosome, administration of K2-6 ribosome, right 4th stage: 2 days after administration of K3 ribosome Administration of ribosome without sugar chain, administration of K3-3 ribosome, administration of K3-4 ribosome, administration of K3-6 ribosome. The rightmost bar shows the fluorescence signal intensity obtained by imaging. In the color imaging diagram, the upper red color of the bar shows the strongest signal, and the lower the color, the weaker the fluorescent signal. In the black-and-white imaging diagram, the fluorescence signal becomes weaker as the white color above the node becomes stronger and the lower it becomes. Unit: photon count, photon / second (ph / s): Count per second The number of fluorescent signals photon to be transmitted.

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 ribosome administration, right 2 Stage: 1 hour after administration of K3 ribosome, no sugar chain from left side Ribosome administration, K3-4 ribosome administration, K3-6 ribosome administration, left 3rd stage: K1 liposome administration 1 day later, left side force is also sugar chain Ribosome administration, K1-3 ribosome administration, K1-4 liposome administration, K1-6 ribosome administration, unadministration, middle 3rd stage: K2 ribosome administration 1 day later, left side force is also glycoside-free ribosome administration, K2-3 ribosome Administration, K2—4 ribosome administration, K2—6 ribosome administration, not administered, right third stage: K3 ribosome administration 1 day later, no glycans from the left side, K3—3 ribosome administration, K3—4 ribosome administration, K3— 6 Liposome administration, no administration, left 4th stage: K1 ribosome administration 2 days later, left side force No sugar chain ribosome administration, K1-3 ribosome administration, K1-4 ribosome administration, K1-6 ribosome administration, central 4th stage Eye: 2 days after administration of K2 ribosome, administration of ribosome without sugar chain, administration of K2-3 ribosome, administration of K2-4 ribosome, administration of K2-6 ribosome, right 4th stage: 2 days after administration of K3 ribosome Administration of ribosome without sugar chain, administration of K3-3 ribosome, administration of K3-4 ribosome, administration of K3-6 ribosome. The rightmost bar shows the fluorescence signal intensity obtained by imaging. In the color imaging diagram, the upper red color of the bar shows the strongest signal, and the lower the color, the weaker the fluorescent signal. In the black-and-white imaging diagram, 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] 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 ribosome administration, K2-6 ribosome administration, right 2 Stage: 1 hour after administration of K3 ribosome, no sugar chain from left side Ribosome administration, K3-4 ribosome administration, K3-6 ribosome administration, left 3rd stage: K1 liposome administration 1 day later, left side force is also sugar chain Ribosome administration, K1-3 ribosome administration, K1-4 liposome administration, K1-6 ribosome administration, unadministration, middle 3rd stage: K2 ribosome administration 1 day later, left side force is also glycoside-free ribosome administration, K2-3 ribosome Administration, K2—4 ribosome administration, K2—6 ribosome administration, not administered, right third stage: K3 ribosome administration 1 day later, no glycans from the left side, K3—3 ribosome administration, K3—4 ribosome administration, K3— 6 Liposome administration, no administration, left 4th stage: K1 ribosome administration 2 days later, left side force No sugar chain ribosome administration, K1-3 ribosome administration, K1-4 ribosome administration, K1-6 ribosome administration, central 4th stage Eye: 2 days after administration of K2 ribosome, administration of ribosome without sugar chain, administration of K2-3 ribosome, administration of K2-4 ribosome, administration of K2-6 ribosome, right 4th stage: 2 days after administration of K3 ribosome Administration of ribosome without sugar chain, administration of K3-3 ribosome, administration of K3-4 ribosome, administration of K3-6 ribosome. The rightmost bar shows the fluorescence signal intensity obtained by imaging. In the color imaging diagram, the upper red color of the bar shows the strongest signal, and the lower the color, the weaker the fluorescent signal. In the black-and-white imaging diagram, 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, K

2-6 ribosome administration, right 2nd row: 1 hour after K3 ribosome administration, left side force is also without liposomes, K3-4 ribosome administration, K3-6 ribosome administration, left 3rd row: K1 ribosome administration 1 One day later, left-side force is also administered without glycans, K1-3 ribosome administration, K1-4 liposome administration, K1-6 ribosome administration, not administered, middle third stage: K2 ribosome administration 1 day later, no glycans from left side Ribosome administration, K2-3 ribosome administration, K2-4 ribosome administration, K2-6 ribosome administration, no administration, right third stage: K3 ribosome administration 1 day later, no sugar chain from left side ribosome administration, K3-3 ribosome administration, K3 — 4 ribosome administration, K3—6 ribosome administration, non-administration, left 4th stage: K1 ribosome administration 2 days later, left side force is also without glycans, K1—3 ribosome administration, K1—4 ribosome administration, K1—6 riboso Administration, middle 4th stage: 2 days after administration of K2 ribosome, ribosome without sugar chain administration from the left side, administration of K2-3 ribosome, administration of K2-4 ribosome, administration of K2-6 ribosome, right 4th stage: 2 days after administration of K3 ribosome, Administration of ribosome without sugar chain from the left side, administration of K3-3 ribosome, administration of K3-4 ribosome, K

3-6 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. In the black-and-white imaging diagram, 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: Κ2 ribosome administration 1 After time, administration of ribosome without sugar chain from the left side, Κ2-4 ribosome administration, Κ2-6 ribosome administration, right second stage: Κ3 ribosome administration 1 hour later, no sugar chain from the left side ribosome administration, Κ3-4 ribosome administration, Κ3-6 ribosome administration, left 3rd stage: K1 liposomal administration 1 day later, left side force is also glycosomal without glycans, K1-3 ribosome administration, K1-4 ribosome administration, K1-6 ribosome administration, unadministered, center 3rd stage: Κ2 ribosome administration 1 day later, left side force is also without glycans, Κ2-3 ribosome administration, Κ2-4 ribosome administration, Κ2-6 ribosome administration, unadministered, right 3rd stage: Κ3 riboso 1 day after administration, no glycans from left side, ribosome administration, Κ3-3 ribosome administration, Κ3-4 ribosome administration, Κ3-6 liposome administration, no administration, left 4th stage: K1 ribosome administration 2 days, left side sugar chain None Ribosome administration, K1—3 ribosome administration, K1—4 ribosome administration, K1—6 ribosome administration, middle 4th stage: Κ2 ribosome administration 2 days later, glycoside-free ribosome administration from the left side, Κ2-3 ribosome administration, Κ2 — 4 ribosome administration, Κ2—6 ribosome administration, right 4th stage: Κ3 ribosome administration 2 days later, left side force is also glycoside-free ribosome administration, Κ3-3 ribosome administration, Κ3-4 ribosome administration, Κ3-6 ribosome administration. The rightmost bar shows the fluorescence signal intensity obtained by imaging. In the color imaging diagram, the upper red color of the bar shows the strongest signal, and the lower the color, the weaker the fluorescent signal. In the black-and-white imaging diagram, 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, Κ2-4 ribosome administration, Κ2-6 ribosome administration, right 2nd stage: Κ3 ribosome administration 1 hour later, no glycan administration from the left side, Κ3— 4 ribosome administration, Κ3-6 ribosome administration, left 3rd stage: K1 liposome administration 1 day later, left side force also without glycans, K1-3 ribosome administration, K1-4 liposome administration, K1-6 ribosome administration, No administration, middle 3rd stage: Κ2 ribosome administration 1 day later, left side force also glycosome without glycan administration, Κ2-3 ribosome administration, Κ2-4 ribosome administration, Κ2-6 ribosome administration, no administration, right 3rd step: Κ3 1 day after ribosome administration, ribosome administration without sugar chain from left side, Κ3-3 ribosome administration, Κ3-4 ribosome administration, Κ3-6 liposome administration, no administration, left 4th stage: K1 ribosome administration 2 days later Left-side force No-glycan ribosome administration, K1-3 ribosome administration, K1-4 ribosome administration, K1-6 ribosome administration, center 4th stage: Κ2 ribosome administration 2 days later, glycoside-free ribosome administration from the left side, Κ2-3 ribosome Administration, Κ2-4 ribosome administration, Κ2-6 ribosome administration, right 4th stage: Κ3 ribosome administration 2 days later, left side force is also without glycans, Κ3-3 ribosome administration, Κ3-4 ribosome administration, Κ3-6 Ribosome administration. The rightmost bar shows the fluorescence signal intensity obtained by imaging. In the color imaging diagram, the upper red color of the bar shows the strongest signal, and the lower the color, the weaker the fluorescent signal. In the black-and-white imaging diagram, 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. 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. In the black and white imaging diagram, 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. In the color imaging diagram, the red color at the top of the bar indicates the lower the color with the strongest signal, the weaker the fluorescent signal. In the black-and-white imaging diagram, 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. In the black and white imaging diagram, the whiter the bar, the stronger the fluorescent signal, and the lower the bar, the weaker the fluorescent signal. 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. In the color imaging diagram, the red color at the top of the bar indicates the lower the color with the strongest signal, the weaker the fluorescent signal. In the black-and-white imaging diagram, 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. In the black and white imaging diagram, the whiter the bar, the stronger the fluorescent signal, and the lower the bar, the weaker the fluorescent signal. 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. Third 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. In the color imaging diagram, the red color at the top of the bar indicates the lower the color with the strongest signal, the weaker the fluorescent signal. In the black-and-white imaging diagram, 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: ¾: 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. HSA, BS,

Three

Tris, DTSSP and SLX are the following abbreviations. HSA; human serum albumin, BS; bis (sulfosuccinimidyl) suberate group, Tris; tris (hydroxymethyl) a

Three

Minomethane group, DTSSP; 3, 3, monodithiopis (sulfosuccinimidyl propionate), S LX; Siaryl Lewis X group.

[FIG. 41] 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; ζΐΖ mice). The inflamed area (back of the hind paw) was observed 24 hours after injection. Measured by eXpl ore Optix (Ex: 680 nm, Em: 700 nm). Data were normalized. 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 (μg / ml) of the reaction mixture when the sugar chain is bound to the liposome surface. (ΌΙ Ο μg / ml, Ό2: 50 μg Zml, D3: 100 μg / ml, D4: 200 μg / ml, D5: 500 μg / ml). The data was entered correctly.

[FIG. 46] 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. The same mouse area of inflammation (back of the hind paw) was observed at 10 minutes, 30 minutes, 6 hours, 24 hours and 48 hours after injection. To stain leukocytes and vascular endothelial cells in the bloodstream, 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. explore Optix (Ex: 680nm , Em: 700 nm). Data was normalized.

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. To stain leukocytes and vascular endothelial cells in the bloodstream, 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). . Six hours after administration, tumor-bearing mice to which Cy7-encapsulated sugar chain-modified ribosome (K-1) was administered had higher fluorescence intensity at the tumor site than the control. At 24 hours after administration, almost no signal was detected.

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.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described below with reference to the best mode. Throughout this specification, it should be understood that the singular forms also include the plural concept unless otherwise stated. It is. Therefore, it should be understood that singular articles (eg, “a”, “an”, “the”, etc. in the English language) also include the plural concept unless otherwise stated. In addition, it should be understood that the terms used in the present specification are used in the meaning normally used in the above field unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

[0023] It is understood that the embodiments provided below are provided for a better understanding of the present invention, and the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention in consideration of the description in the present specification.

[0024] Definitions of terms particularly used in the present specification will be described below as appropriate.

In the present specification, 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. Examples of such sugar chains 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”. Further, unless otherwise specified, in this specification, “sugar chain” may include both sugar chains and sugar chain-containing substances. Typically about 20 monosaccharides (such as dalcoose, galactose, mannose, fucose, xylose, N-acetylyldarcosamine, N-acetylgalatatosamine, sialic acid and their complexes and derivatives) Is a chain-linked substance that is attached to proteins and lipids inside and outside the body of cells. Functions differ depending on the arrangement of monosaccharides, and they are usually branched in a complex manner. 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. It is believed that there are over 10,000 types. It seems to be related to higher-order functions that proteins and lipids perform in vivo, such as intercellular molecular / cell recognition functions, but the mechanism is still unclear. It is attracting attention in the current life science as the third life chain after nucleic acid and protein. In particular, the function of a sugar chain as a ligand (information molecule) in cell recognition is expected, and its application to the development of highly functional materials is being studied.

In this specification, “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. For example, examples of the sugar chain group include sialyl Lewis X group, N-acetylyl lactosamine group, and α 1-6 mannobiose group.

In the present specification, “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. In this specification, sugar is also referred to as a carbohydrate, and both are used interchangeably. In the present specification, when specifically mentioned, 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.

Where η = 2, 3, 4, 5, 6, 7, 8, 9 and 10 are diose, triose, tetrose, pentose, hexose, heptose, otatose, nonose and decouse, respectively. That's it. It is generally equivalent to an aldehyde or ketone of a chain polyhydric alcohol. The former is called aldose and the latter is called ketose. In the present invention, any type can be used.

[0028] The nomenclature and abbreviations used to describe sugars in the present invention follow normal nomenclature. For example, β D galactose

[0029] [Chemical 11]

β-D-Galactose

The Gal;

N Acetinore a D Galactosamine [0030] [Chemical 12]

N-Acety [-aD-galactosamine

The GalNAc;

D Manno

[0031] [Chemical 13]

α-D-Mannose

pD-Glucose

Glc;

N acetyleno β D darcosamine

[0033] [Chemical 15]

N-Acetvl-pD-glucosamine

GlcNAc;

: L course

[0034] [Chemical 16]

Fuc;

a—N acetylneuraminic acid

[Chemical 17]

α-Ν-Acet l neuraminic acid

Is Neu5Ac;

Ceramide

[0036] [Chemical 18]

Ceramide

Cer;

L-Serine

CH (OH) CH (COOH) NH

twenty two

Is represented by Ser. Cer 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. In addition, Ser is usually classified as an amino acid. In this specification, 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 ひ and j8. It may be expressed as a or b for display reasons. Therefore, in the present specification, α and a, j8 and b are used interchangeably for the anomeric notation.

[0037] As used herein, galactose refers to any isomer, but typically 13 D It is galactose and is used to refer to j8-D-galactose unless otherwise stated.

[0038] In this specification, 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.

In the present specification, mannose refers to any isomer, but is typically a-D-mannose, and is used to refer to ex D-mannose unless otherwise specified.

[0040] In the present specification, glucose refers to any isomer, typically j8-D.

Glucose, unless otherwise mentioned, is used to refer to 13 D-glucose.

[0041] As used herein, acetylyldarcosamine refers to any isomer, but is typically N-acetylenic β D darcosamine, and unless otherwise specified, refers to ァ acetylene β-D-darcosamine. Used as a thing.

In the present specification, fucose refers to any isomer, but is typically a L-fucose, and is used to refer to α L-fucose unless otherwise specified.

[0043] In the present specification, acetyl-neuraminic acid refers to any isomer, but is typically α-Ν acetylneuraminic acid, and α-Ν-acetylenuraminic acid is referred to unless otherwise specified. Used as a pointer.

[0044] In this specification, serine refers to any isomer, but is typically L-serine, and is used to refer to L-serine unless otherwise specified.

[0045] In the present specification, it should be noted that the symbol, designation, abbreviation (Glc, etc.), etc. of sugars are different from those used when representing a monosaccharide and when used in a sugar chain. In the sugar chain, the unit sugar has a dehydration condensation with another unit sugar to which it is bonded, so that the mutual force also exists in a form excluding hydrogen or hydroxyl group. Therefore, when these abbreviations for sugars are used to represent monosaccharides, all hydroxyl groups are present, but when used in a sugar chain, the hydroxyl groups are bound to the hydroxyl groups of the sugars to which they are attached. It is understood that only oxygen remains after dehydration condensation Is done.

[0046] When a sugar is covalently bonded to albumin, the reducing end of the sugar is aminated, and the ability to bind to other components such as albumin via the amino group. In that case, the reducing end hydroxyl group Note that refers to those substituted with amine groups.

[0047] 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 α and j8. Also described are specific carbon atoms that form a bond between two carbons.

[0048] In the present specification, the sugar chain is

[0049] [Chemical Formula 19] A monosaccharide is represented by an anomaly-like A monosaccharide. Thus, for example, the e-glycosidic bond between C-1 in galactose and C-4 in glucose is represented by Gal | 8 1,4Glc. Thus, for example, Siaryl Lewis X (SLX) is represented as Neu5Ac a 2,3Gal β 1,4 (Fuc α 1,3) GlcNAc. N-acetyllactosamine (G4GN) is represented as Gal jS 1, 4GlcNAc. α 1-6 Mannobiose (A6) is expressed as Man a 1, 6Man.

[0050] Branches of sugar chains are represented by parentheses, and are arranged immediately to the left of the unit sugar to be bound. For example,

[0051] [Chemical Formula 20] Monosaccharide Anomer £ is expressed as a simple sugar,

[Chemical Formula 21] Monosaccharide anomeric ι = Like A Thus, for example, if a galactose C 1 and a glucose C 4 have a | 8 glycosidic bond, and the glucose C-3 has an α-glycoside bond with a fucose C-1 then Gal jS 1, 4 It is expressed as (Fuc α 1, 3) Glc. Monosaccharides are represented on the basis of the lowest possible number of (latent) carbo groups. Of organic chemistry nomenclature In general standards, even when a group superior to a (latent) carbo group is introduced into a molecule, it is usually represented by the above numbering.

[0053] [Chemical 22]

[0054] [Chemical 23]

[0055] Examples of the sugar chain used in the present specification include sugar chains selected from the group having sialyl Lewis X, N-acetyllactosamine, α 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.

[0056] (Ribosome)

In the present specification, the “ribosome” usually means a closed vesicle composed of a lipid layer assembled in a film form and an inner aqueous layer. In addition to 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. In the present invention, the ribosome imparts a structural unit having a functional group that imparts an ester bond (for example, a glycolipid, gandioside, 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).

[0057] 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. a) Periodate oxidation of the gandioside moiety on the ribosome membrane, and b) Coupling of the glycoprotein to the oxidized ribosome by a reductive amino acid reaction. An example of the reaction flow is shown in Fig. 31. By such a technique, 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, gandioside 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.

[0058] 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.

[0059] Examples of phosphatidylcholines include dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, and the like.

[0060] Examples of phosphatidylethanolamines include dimyristoyl phosphatidylethanol. And amine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine and the like.

[0061] Examples of phosphatidic acids include dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, and distearoyl phosphatidic acid. Examples of long-chain alkyl phosphates include dicetyl phosphate.

[0062] Examples of the glycolipids include galactosylceramide, darcosylceramide, latatosylceramide, phosphatide, globoside, and gandiosides. 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.

[0063] As the phosphatidylglycerols, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol and the like are preferable.

[0064] Of these, 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. For example, as the lipid constituting the ribosome of the present invention, 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%) And those containing cholesterol (molar ratio 0 to 70%). Preferably, containing gandarioside, glycolipid or phosphatidylglycerol. This is because the binding of a linker such as albumin becomes easy.

[0065] Preferably, according to the embodiment, 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. .

[0066] When the ribosome is produced, 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. [0067] In another preferred embodiment, it is preferred that 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).

[0068] (Sugar chain-modified ribosome)

In one aspect, the present invention provides 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. In a specific embodiment, 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, α 1-6 mannobiose, and a combination force thereof. Are connected.

[0069] As used herein, the term "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. One hour. Specifically describing the form of sugar chains bound to ribosomes,

Structure IX-R ¹ -R ² -R ³

X: The linker protein contained in the ribosome can bind to CH—ΝΗ

2

A group in which the functional group a is removed from the structural unit containing the functional group a

R ¹ : Linker protein group

R ² : Linker-protein cross-linking group

R ³纖

X and R ¹ are CH—NH bonded, R ¹ and R ² are peptide bonded, and R ² and R ³ are peptide

2

Are connected.

Thus, in more detail, structure I is

X-CH NH— R ¹ — NH— C (= 0) — R ² — C (= 0) — NH— R ³

2

t can be represented by a structural formula.

[0070] 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, and R ⁴ and R ⁵ are peptide bonds.

Thus, in more detail, structure II is

Y-NH-C (= 0)-R ⁴ -C (= 0) -NH-R ⁵

t can be represented by a structural formula.

[0071] In one embodiment, the ribosome of the present invention has the above-described Structure I and Structure II, and is fluorescent.

[0072] In the present invention, 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).

[0073] 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.). As the fluorescent dye, for example, cy5.5 (for example,

[0074] [Chemical 24]

<Cy5. 5>

1, 1'-bis (ε-carboxypentyl) -3, 3, 3 ', 3'-tetramethyl-3Η-benzindodicarbocyanine 5,5', 7, 7'-tetrasulfonate Potassium salt Roxisuccinimide ester),

cy3 (for example,

[Chemical 25]

<cy3>

, 1 (ε carboxypentyl) 1'-ethyl 3, 3, 3 ', 3, -tetramethylindocarbocyanin 1,5,1 disulfonate potassium salt 1-hydroxysuccinimide ester (cy3) etc.), cy5, cy7, cy3B, cy3.5, 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.

As used herein, “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. For example, 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. [0077] The sugar chain-modified ribosomes used herein can be included at a density suitable for delivery to the intended delivery site.

[0078] As used herein, "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). Although 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.

[0079] [Table 1]

(table 1 )

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. A step of producing a bound ribosome; and (f) a step of binding a sugar chain described in the above table to the liposome to produce a sugar chain-modified ribosome. [0081] Preferably, in this method, the fluorescent labeling solution in step (c) is 1, 1'-bis (ε-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.

[0082] The 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.

[0083] The sugar chain-modified ribosome of the present invention can encapsulate or bind a drug or gene. Examples of the drugs include alkyl 匕 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-cytokinin receptors' chemokine receptor antibodies, siRNA, miRNA, smRNA, antisense oligodeoxynucleotide (ODN) or DNA therapy-related nucleic acid preparations such as DNA, neuroprotective factors, antibodies Drugs, molecular targeted drugs, osteoporosis / bone metabolism-improving drugs, neuropeptides, bioactive peptides / proteins, but not limited to these.

[0084] As used herein, "linker" is a molecule that mediates the binding between a sugar chain and the ribosome surface. In the sugar chain-modified ribosome of the present invention, 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. In the present specification, 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.

V, confirmed by previous experiments!

In the present specification, 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!

In the present specification, 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. In the present specification, 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.

In this specification, “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. Examples of linker protein crosslinking groups that can be used herein 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.

[0088] As used herein, 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

[0089] As used herein, "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.

[0090] As used herein, "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.))). Preferably, 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.

[0091] As used herein, "human-derived serum protein" refers to a protein contained in a liquid portion that remains when human blood naturally coagulates. In this specification, when a human-derived protein shows a state of being bound to another substance, it is referred to as a human-derived protein group.

[0092] As used herein, "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.

[0093] In the sugar chain-modified ribosome according to the present invention, at least one of the ribosome membrane and the linker is hydrophilicized by binding a hydrophilic compound, preferably, a tris (hydroxyalkyl) aminoalkane. Moyo.

[0094] As used herein, "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. 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. In addition, 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.

As used herein, “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

It can be linear or branched. In the present specification, the “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. For 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 alkane. Here, for example, C1-C10 alkyl means linear or branched alkyl having 1 to 10 carbon atoms, and methyl (CH—)

Three

, Ethyl (C H —), n-propyl (CH 2 CH 2 CH 1), isopropyl ((CH 2) CH—)

2 5 3 2 2 3 2

, N-butyl (CH CH CH CH 1), n-pentyl (CH CH CH CH CH —), n

3 2 2 2 3 2 2 2 2 Monohexyl (CH CH CH CH CH CH One), n-heptyl (CH CH CH CH CH

3 2 2 2 2 2 3 2 2 2

CH CH 1), n-year-old cutinore (CH CH CH CH CH CH CH CH 1), n-noninore ( CH CH CH CH CH CH CH CH CH 1), n-decyl (CH CH CH CH CH

3 2 2 2 2 2 2 2 2 3 2 2 2 2

CH CH CH CH CH 1), 1 C (CH) CH CH CH (CH), 1 CH CH (CH)

Examples are 2 2 2 2 2 3 2 2 2 3 2 2 3 2 and the like. In addition, for example, C1-C10 substituted alkyl refers to C1-C10 alkyl, in which one or more hydrogen atoms are substituted with a substituent. As R, C1-C6 alkyl is preferred, and C1-C6 alkyl is particularly preferred.

[0096] When the substance of the present invention or the functional group defined above is substituted by a substituent R, such 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.

[0097] Furthermore, 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. For example, 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.

[0098] ZR ¹ (R ² OH) Formula (1)

H NR ^3- (R ⁴ OH) Formula (2)

2 n

H NR ⁵ (OH) Formula (3)

2 n

Where RR ³ and R ⁵ are C force C, preferably C to C, more preferably C force

1 40 1 20 1 to C represents a straight or branched hydrocarbon chain, and R ² and R ⁴ are absent or C

10 1 et al C, preferably C-force C, more preferably C-force C linear or branched carbonization

40 1 20 1 10

Indicates a hydrogen chain. 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

2

Imide, imide ester, active halogen, EDC, pyridyl disulfide, azido file, hydrogen And dolazide. 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. However, since 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.

In the present specification, the “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.

[0100] In the present specification, 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. Examples of 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. Preferably, the hydrophilic compound crosslinking group is a bis (sulfosuccinimidyl) suberate group.

[0101] 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 trehalose solution were used, and a crude dispersion of multilayer ribosomes was prepared by the method of Bangham et al. (See J. Mol. Biol. 8, 660-668 (1964)). It can also be done by adopting a method such as;)). Of these, Tris (hydroxy It is particularly preferred to make the ribosome surface hydrophilic with methyl) aminomethane. 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. For example, in the case where a sugar chain is bound on a ribosome and its molecular recognition function is used for target orientation as in the present invention, tris (hydroxymethyl) aminomethane is a low molecular weight substance, so that conventional polyethylene glycol or the like can be used. Compared to the method using a high molecular weight substance, it is a steric hindrance to the sugar chain and is particularly preferred because it does not prevent the progress of the sugar chain molecule recognition reaction by the lectin (sugar chain recognition protein) on the target cell membrane surface.

[0102] Further, 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. Bissulfosuccinimidyl suberate, disuccinimidyl glutarate, dithiobis succinimidinorepropionate, disuccinimidino resberate, 3, 3'-dithiopis (sunophosxin Imidyl propionate), ethylene glycol bissuccinimidyl succinate, ethylene glycol bissulfosuccinimidyl succinate, etc. 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.

[0103] Thus, 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.

[0104] As used herein, "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 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. There is a method using a carrier to which an antibody or a sugar chain is bound, and it is sometimes called a “missile drug”.

[0105] As used herein, "drug delivery vehicle" refers to a vehicle for delivering a desired drug.

[0106] In another aspect, 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. Typically, 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. For example, a suitable vehicle can be water for injection, physiological solution, or artificial cerebrospinal fluid.

[0107] 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, α-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).

[0108] Exemplary suitable carriers include neutral buffered saline or saline mixed with serum albumin. Preferably, the product is formulated as a lyophilizer using a 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.

[0109] In one aspect, 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.

[0110] As used herein, 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.

[0111] 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.

[0112] 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. By binding both glycans that enhance intestinal absorption control and sugar chains that are directed to specific tissues or organs to ribosomes, the characteristics of both directivity to specific tissues or organs and intestinal absorption control can be achieved. It is also possible to produce ribosomes that have both. [0113] In the sugar chain-modified ribosome contained in the imaging agent of the present invention, 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. OOOlmg sugar chain / mg lipid to 500 mg sugar chain / mg lipid. When imaging inflammation, it is preferable that 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. When imaging cancer tissue, 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.

[0114] In the sugar chain-modified ribosome contained in the imaging agent of the present invention, 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. When imaging cerebral blood vessels, 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). obtain. When imaging the liver, it is preferred that 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). Can be included

[0115] In the sugar chain-modified ribosome contained in the imaging agent of the present invention, the sugar chain may be an a 1-6 mannobiose group. This α 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. When imaging cancer tissue, 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] In one aspect, the present invention provides a carrier for use in molecular or in vivo imaging. This carrier may include a sugar chain-modified ribosome.

[0123] In the sugar chain-modified ribosome contained in the carrier of the present invention, 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. When imaging inflammation, 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) . 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.

[0124] In the sugar chain-modified ribosome contained in the carrier of the present invention, 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. 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). Can be. When imaging the liver, 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). .

[0125] In the sugar chain-modified ribosome contained in the carrier of the present invention, the sugar chain may be a 1-6 mannobiose group. This α 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. 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 sugar chain Z mg lipid (preferably 0.025 mg sugar chain Z mg lipid). obtain.

[0126] 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.

[0127] 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). Say. Thus, 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.

[0128] 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

50

And LD, doses that are lethal to 50% of the population). Therapeutic effect

50

The dose ratio between fruit and toxic effects is the therapeutic index, expressed as the ratio ED ZLD.

50 50 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,

50

It will vary within this range depending on the sensitivity of the patient and the route of administration. As an example, the dose is appropriately selected depending on age and other patient conditions, the type of disease, the type of cells used, and the like.

[0129] The 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.).

[0130] 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. In the sugar chain-modified ribosome contained in the composition of the present invention, 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. When imaging inflammation, it may preferably be included in a glycosylated liposome with a modified binding 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). . When imaging cancer tissue, 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). .

[0131] 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). Can be. When imaging the liver, 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). .

[0132] In the sugar chain-modified ribosome contained in the composition of the present invention, the sugar chain may be an a 1-6 mannobiose group. This α 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. 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 sugar chain Z mg lipid (preferably 0.025 mg sugar chain Z mg lipid). obtain.

[0133] 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.

[0134] In the present specification, 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.

[0135] As used herein, "subject" refers to an organism to which the treatment of the present invention is applied, and is also referred to as "patient". The patient or subject may preferably be a human.

[0136] (Use of sugar chain-modified ribosome as a medicine) In another aspect, 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. Provide the use of modified ribosomes. Here, as the sugar chain-modified ribosome, any form described in the above (Sugar chain-modified ribosome) can be used.

[0137] In another aspect, the present invention can also be used as a medicament. In such a case, 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 Medicine, blood Gene therapy-related nucleic acids such as anti-neoplastic agents, cytoforce-in, chemokines, anti-site-force-in antibodies, anti-chemokine antibodies, anti-site-force-in chemokine receptor antibodies, siRNA, miRNA, smRNA, antisense ODN or DNA Drugs such as pharmaceuticals, neuroprotective factors, antibody drugs, molecular targeted drugs, osteoporosis, bone metabolism improving drugs, neuropeptides, bioactive peptides' proteins.

[0138] (Imaging method)

In another aspect, 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. To do. Here, as the sugar chain-modified ribosome, any form described in the above-mentioned (sugar chain-modified liposome) can be used.

[0139] In another aspect, 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. Here, as the sugar chain-modified ribosome, any form described in the above (Sugar chain-modified ribosome) can be used.

[0140] (Imaging system)

In another aspect, the present invention provides a system for molecular imaging or in vivo imaging of a site of interest. As used herein, “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

A) a sugar chain-modified ribosome specific to the target site;

B) sign; and

C) means for examining the presence or absence of the label;

Where, where

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 can be imaged by the label. The label includes, but is not limited to, a fluorescent substance, a radioactive substance, a coloring substance (for example, β gal), an issuing substance (for example, luciferase), and the like.

[0141] 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. When imaging inflammation, 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). obtain. When imaging cancer tissue, 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). .

[0142] 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. When imaging cerebral blood vessels, preferably, 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). When imaging the liver, 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). .

[0143] 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 α 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. 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.

[0144] With the system of the present invention, 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. Preferably, the means for checking the presence or absence of the label further comprises a stick objective lens. As a means for examining the presence or absence of a label, any substance (for example, fluorescence, radiation, chromogenic substance (for example, βgal), issuing substance (for example, luciferase), etc.) can be detected as long as the label can be detected. Etc.). This is because the function or structure of the living body can be imaged by detecting the label.

[0145] (Treatment method)

In another aspect, 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. Here, as the sugar chain-modified ribosome, any form described in the above (Sugar chain-modified ribosome) can be used.

[0146] In another aspect, 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. Here, as the sugar chain-modified ribosome, any form described in the above-mentioned (Sugar chain-modified ribosome) can be used.

[0147] (Delivery method)

In another aspect, 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. Here, as the sugar chain-modified ribosome, any form described in the above (sugar chain-modified liposome) can be used. Here, as the sugar chain-modified ribosome, any form described in the above (Sugar chain-modified ribosome) can be used.

[0148] (Production method)

In one aspect, 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. Here, as the sugar chain-modified ribosome, any form described in the above-mentioned (Sugar chain-modified ribosome) can be used.

[0149] In another aspect, the present invention provides a method for producing a sugar chain-modified ribosome. In this method, (a) 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. And (f) binding a sugar chain to the ribosome to produce a sugar chain-modified ribosome. Here, as the sugar chain-modified ribosome, any form described in the above (Sugar chain-modified ribosome) can be used. [0150] In another aspect, 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. 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 (eg, fluorescent dyes, pharmaceuticals) can include, for example, encapsulation, binding to the outer surface, and the like. Here, as the sugar chain-modified ribosome, any form described in the above (Sugar chain-modified ribosome) can be used.

[0151] (Bind or encapsulate fluorescent dye)

For the binding or encapsulation of the fluorescent dye, any of those used to bind or encapsulate drugs in ribosomes is used. For example, (1) dinormitoylphosphatidylcholine (DPPC), cholesterol, gandarioside, dicetylphosphate (DCP), dipalmitoylphosphatidylethanolamine (DPPE), 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. Resuspended in N-tris (hydroxymethyl) 3-aminopropanesulfonate buffer (pH 8.4), stirred at 37 ° C for 1 hour, purged with nitrogen, and sonicated. (3) Ultrasonic Mix the treatment solution and fluorescent dye (eg, cy5.5, cy3, cy7) labeled human serum albumin solution (fluorescent dye labeled HSA solution) and perform ultrafiltration (fractional molecular weight: 10,000). To obtain a ribosome particle encapsulating a fluorescent dye (eg, cy5.5, cy3, cy7) labeled HSA.

[0152] (Production of ribosome)

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. Prepare mixed micelles of lipid and surfactant sodium cholate. In particular, the combination of phosphatidic acids or long-chain alkyl phosphates such as dicetyl phosphate is essential to negatively charge the ribosome, and the combination of phosphatidylethanolamines is a hydrophilic reaction site. Formulation of gandiosides or glycolipids or phosphatidylglycerols is essential as a binding site for the linker. Group power consisting of gandiosides, 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.

[0154] The present invention also includes liposome itself by binding sugar chains that have been hydrophilicized using the above-mentioned hydrophilic compound. Such 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%).

[0155] 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.

[0156] Preferably, the hydrophilic property can be performed as follows. (1) Carbonate buffer (50mM

NaHCO, 157 mM NaCl

3. Perform ultrafiltration (molecular weight cut off: 300,000) for buffer exchange to pH 8.5). (2) Add the cross-linking agent BS ³ (PIERCE), stir at room temperature for 2 hours, and then stir overnight under refrigeration. (3) Perform ultrafiltration (molecular weight cut off: 300, 000) to remove free BS ³ . (4) Add tris (hydroxymethyl) aminomethane Z carbonate buffer (PH8.5) solution, stir at room temperature for 2 hours and then refrigerate overnight. (5) Ultrafiltration (molecular weight cut off: 300, 000) to remove free tris (hydroxymethyl) aminomethane and exchange the buffer with N-tris (hydroxymethyl) 3-aminopropanesulfonic acid buffer (pH 8.4). )do.

[0157] More preferably, 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)),

3,

Perform ultrafiltration twice (fractionated molecular weight: 100,000, 2000 X g for 60 minutes). (2) Add cross-linking agent BS ³ (PIERCE), and after 2 hours at room temperature, stir overnight under refrigeration. (3) Perform ultrafiltration (fractionated molecular weight: 100,000, 2000 X g for 60 minutes) twice to remove free BS ³ (4) Add tris (hydroxymethyl) aminomethane / CBS buffer (pH 8.5) solution, and after 2 hours at room temperature, stir overnight under refrigeration. (5) Ultrafiltration (fractionated molecular weight) to remove free tris (hydroxymethyl) aminomethane and exchange the buffer with N-tris (hydroxymethyl) 3-aminopropanesulfonate buffer solution (PH8.4) : 100,000, 2000 X g for 60 minutes) twice.

[0158] In one embodiment, 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. Here, as the sugar chain-modified ribosome, any form described in the above-mentioned (Sugar chain-modified ribosome) can be used.

[0159] In a preferred embodiment, 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).

[0160] In one embodiment, 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). And b) concentrating the solution containing the fluorescently encapsulated ribosome and adding the solution A to the concentrated solution containing the fluorescently encapsulated ribosome; and (b2) the solution A The mixed solution containing the fluorescently encapsulated ribosome to which is added a fractional molecular weight of 300,000 And c) concentrating, concentrating, and adding the solution A to the concentrated mixed solution to prepare the fluorescently encapsulated ribosome that has been made hydrophilic. (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. (Preferably 37 ° C) for 3-7 days (preferably 3 days) and incubate under refrigeration (eg about 2-8 ° C) for 20-60 minutes (preferably 30 minutes) And a filtration filter to prepare an aminated sugar chain solution; (d3) adding the aminosaccharide chain solution to the solution containing the hydrophilically encapsulated fluorescent encapsulated ribosome and mixing Shi After, at room temperature (e.g., about 15 to 30 ° C, preferably in the range of 20-25 ° C is) 2-6 hours (good Preferably, the reaction solution step obtained by reacting for 2 hours; and (d4) solution B containing Tris buffer is added to the reaction solution obtained by step (d3), and room temperature (for example, about 15 -30 ° C, preferably 20-25 ° C) and stirred for 2-6 hours (preferably 2 hours) and refrigerated (eg 1-10 ° C, preferably 2-8 ° C) under stirring for 16 to 48 hours (preferably 16 to 20 hours), ultrafiltered with a molecular weight cut off of 30,000, and desalted to produce the fluorescent dye-containing sugar chain-modified ribosome. 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. When the primary amino group already exists in the sugar chain, the step of preparing the aminated sugar chain solution of (d 2) can be omitted. When carrying out the amination reaction, 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. Say.

[0161] 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.

[0162] The sugar chains that can be used in the present method include, but are not limited to, sialic Lewis X, N-acetylyl lactosamine, α 1-6 mannobiose, and the like. The sugar chain may preferably be Cialyl Lewis X.

[0163] In another embodiment, the sugar chain that can be used by the method of the present invention can be a sugar chain capable of glycosylation reaction.

[0164] 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. When a sugar chain specific to the inflammatory system is used, the sugar chain can be preferably added at a final concentration of 5 mM.

[0165] (kit for producing sugar chain-modified ribosomes containing fluorescent dyes) In one aspect, 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. Here, as the sugar chain-modified ribosome, any form described in the above (sugar chain-modified ribosome) can be used.

[0166] In another aspect, 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. Here, as the sugar chain-modified ribosome, any form described in the above (sugar chain-modified ribosome) can be used. The Tris buffer used in this kit is a buffer using Tris-hydroxyme thylammonium as a basic component, for example, N tris (hydroxymethyl) -3-amino. Propane sulfonate buffer or the like can be used.

[0167] As used in the present invention, "refrigerated (lower)" refers to a temperature ranging from about 1 ° C to about 12 ° C, preferably from about 2 ° C to about 8 ° C. As used herein, “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.

[0168] (Synthesis of sugar chains)

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). (WO2002 / 081723, JP-A-9 31095, JP-A-11-4 2096, JP-A-2004-180676, Kenichi Hatanaka, Shinichiro Nishimura, Goro Ouchi and Kazuyoshi Kobayashi (1997) Carbohydrate Science and Industry, Kodansha See Tokyo etc.).

[0169] 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)

In the present invention, 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. At this time, 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. In this case, 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. By selecting multiple sugar chains such as the former, it is possible to reliably target a specific target tissue or organ. By selecting multiple sugar chains such as the latter, multiple sugar chains can be selected for one type of ribosome. Target can be directed and multipurpose target-directed ribosomes can be obtained.

[0171] In order to bind the sugar chain to the ribosome, it is possible to mix the linker and / or sugar chain during the production of the ribosome, and to bind the sugar chain to the surface while producing the ribosome. It is desirable to prepare ribosomes, linkers and sugar chains separately, and link the linkers and z or sugar chains to the completed ribosomes. This is because the density of the sugar chain to be bound can be controlled by binding the linker and Z or sugar chain to the liposome. Direct binding of sugar chains to ribosomes can be performed by the methods described below.

[0172] 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. When linking sugar chains using a linker, it is preferable to use 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. For example, the ability of 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.

[0173] In the ribosome of the present invention, 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.

[0174] As described later, when the target-directed ribosome of the present invention is used as a medicine, 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. In this case, the protein may also serve as a linker for binding the ribosome and sugar chain and a protein having a medicinal effect. Examples of the medicinal protein include physiologically active proteins.

[0175] In the production of the sugar chain-modified ribosome of the present invention, a crosslinking group can be used when the ribosome and the linker are bound.

[0176] In order to bind a sugar chain to a ribosome via a linker, the following method may be used.

[0177] First, a protein is bound to the ribosome surface. Ribosome, NalO, Pb (0 CCH

4 2 3

) Gandarioside present on the ribosome membrane surface is treated with an oxidizing agent such as NaBiO.

4 3

And then NaBH CN

3 Using the test drive of NaBH, etc., on the linker and ribosome membrane surface

Four

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. , Dithiobissuccinimidyl propionate, disuccinimidino resverate, 3, 3, 1 dithiobis (sulfosuccinimidyl propionate), ethylene glycolenobisbiscin imidyl succinate, ethylene 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. [0178] Specifically, first, one end of a divalent reagent for crosslinking is bonded to all the amino groups of the linker. Then, 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. For example, when a linker and a sugar chain are covalently bonded via a disulfide bond, the sugar chain is cleaved by reduction in the cell. When the sugar chain is cleaved, the surface of the liposome becomes hydrophobic, binds to the biological membrane, disturbs the membrane stability, and releases the drug contained in the ribosome.

[0179] Next, most of the divalent reagents that bind to the surface of the protein on the sugar chain-modified ribosome membrane thus obtained! Perform hydrophilic treatment using unreacted ends. In other words, the unreacted end of the divalent reagent bound to the protein on the ribosome and the compound used for the above hydrophilic property such as tris (hydroxymethyl) aminomethane are subjected to a binding reaction to make the entire ribosome surface hydrophilic. To do. The hydrophilic property of the ribosome surface and the linker improves transferability to various tissues, retention in blood, and transferability to various yarns and tissues. This is because the surface of the ribosome and the surface of the linker are made hydrophilic, so that the parts other than the sugar chain appear to be the moisture in the body in each tissue. This is probably due to the fact that only the sugar chain is recognized by the target tissue lectin (sugar chain recognition protein).

[0180] Preferably, 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.

[0181] Preferably, 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.

[0182] More preferably, 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. (4) free Xia Nohou sodium periodate to remove the HSA, carbonate buffer _(P H8. 5) in a purpose to buffer exchange, and concentrated to 1 / 5-1 / 10 times by ultrafiltration, Add carbonate buffer (pH 8.5) to the original volume. Repeat this operation twice. This allows the linker to bind to the ribosome.

[0183] More preferably, 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. (4) For the purpose of exchanging the buffer with carbonate buffer (pH 8.5), concentrate to 1Z5 to 1Z10 times by ultrafiltration, and add carbonate buffer (PH8.5) to the original volume. Repeat this operation twice. As a result, the linker can be bound to the ribosome.

[0184] Next, the sugar chain is bound to a linker on the ribosome. For this purpose, the reducing end of the sugar constituting the sugar chain is glycosylated using ammonia salts such as NH HCO and NH COONH.

4 3 2 4

And then bis-sunophossuccinimidinores belate, disuccinimidino regaleate, dithiobissuccinimidyl propionate, disuccinimidino resverate, 3, 3, 1 dithiobis ( Bind on the ribosome membrane surface using a bivalent reagent such as sulfosuccinimidyl propionate), ethyleneglycololebissuccinimidyl succinate, ethylene glycol bissulfosuccinimidyl succinate The obtained linker and the glycosylated amino sugar are bound to obtain a ribosome. These sugar chains are commercially available.

[0185] Preferably, 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. (5) Perform ultrafiltration (molecular weight cut off: 300,000) to remove free sugar chains and tris (hydroxymethyl) aminomethane and exchange the buffer with HEPES buffer (pH 7.2). Thereby, the binding of the sugar chain to the linker on the ribosome can be achieved.

[0186] 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). [0187] 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.

[0188] [Chemical 26] <Principle of BCA method>

Process 1. Protein + GU ⁺ 2

[0189] For the measurement of the amount of protein, for example, Micro BCA Protein Assay Reagent Kit (Cat. No. 23235BN) (PIERCE Co. LTD) can be used. As a standard, 2 mg Zml albumin (BSA) can be used. (1) As a standard solution, dilute the standard substance (2mgZml: albumin) with PBS buffer solution to prepare 0, 0.25, 0.5, 1, 2, 3, 4, 5 g / 50 1 solution. (2) Dilute the Cy5.5-encapsulated sugar chain-modified ribosome 20-fold with PBS buffer to prepare a sample solution. (3) Dispense the standard solution and sample solution into test tubes for 50 1 minutes. (4) Add 100 1 of 3% sodium lauryl sulfate solution (SDS solution) to each test tube. (5) Mix reagents A, B, and C attached to the kit so that reagent A: reagent B: reagent C = 48: 2: 50, and add 1501 to each test tube. (6) Leave the test tube at 60 ° C for 1 hour. (7) After returning to room temperature, measure the absorbance at 540 nm, create a calibration curve with a standard solution, and measure the amount of ribosomal protein. Figure 32 shows an example of a calibration curve.

[0190] 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.

[0191] 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.

[0192] <Principle of lipid quantification>

[0193] [Chemical 27]

Esters ¾ Cholesterol (EC) ¾ Crane Cholesterol (FC) Fatty Acid

Free cholesterol (FC) 厶 Cholestenone Hydrogen peroxide

[0194] For the quantification of lipids, for example, the Detamina TC555 kit (catalog number UCCZEAN12 8) (KYOWA Co. LTD) can be used. Use 50 mg Zml cholesterol attached to the kit as a standard substance. (1) As a standard solution, 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. (2) Dilute Cy5.5-encapsulated sugar chain-modified ribosome 5 times with PBS buffer to prepare a sample solution. (3) Dispense 20 μ1 each of the standard solution and the sample solution into test tubes. (4) Add 1 7 1 TritonX-100 (10% solution) to each test tube, stir, and then allow to stand at 37 ° C for 40 minutes. (5) Add 3001 enzyme reagent of Detamina TC555 Kit, stir, and then let stand at 37 ° C for 20 minutes. (6) Absorbance was measured at 540 nm, and a calibration curve (an example of a calibration line is shown in Figure 33) using a standard solution. . ) To measure the amount of cholesterol in the ribosome and determine the amount of lipid.

[0195] The conversion formula for obtaining the lipid content is also expressed as follows, for example.

Lipid content Ζ50 / ζ 1) = Cholesterol content ₈ Ζ50 / ζ 1) Χ4.51 (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.

[0196] 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.

[0197] 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.

[0198] The ribosome and sugar chain-modified ribosome of the present invention preferably have a particle size of about 80 ηm 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. 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.

[0199] 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. For example, 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, lentinan, schizophyllan, Examples include medroxyprogesterone acetate, phosfestol, mepitiostan, and epitiostanol. In the present invention, the above-mentioned drugs include derivatives thereof.

[0201] By including a drug as described above, the ribosome of the present invention can be used for the treatment of diseases such as cancer and inflammation. Here, cancer includes all neoplastic diseases such as tumor and leukemia. When 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. Compared with the administration of ribosome (reference ribosome) to which tris (hydroxymethyl) aminomethane is bound, it can accumulate 3 to 4 times, preferably 4 to 6 times.

[0202] 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.

[0203] The compound having a medicinal effect may be encapsulated in a ribosome or bound to the surface of a liposome. For example, 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. in ribosomes, a well-known method can be used.For example, solutions containing drugs and phosphatidylcholines, phosphatidylethanolamines, phosphatidic acids or long-chain alkyl phosphates, gangliosides, By forming ribosomes using glycolipids or lipids containing phosphatidylglycerols and cholesterols, drugs and the like are encapsulated in ribosomes. [0204] Therefore, 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. .

[0205] When the molecular imaging agent of the present invention is used for diagnosis, 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.

[0206] (Health 'food)

The present invention can also be used in the health 'food field. In such a case, the points to be noted when used as an oral medicine should be considered as necessary. In particular, when it is used as a functional food such as a specific health food or a “health food”, it is preferable to treat it according to a pharmaceutical. Preferably, 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.

[0207] For example, yew, leaves, saw palmetto, St. John's wort, Norrelian, black cohosh, milk thistle, evening primrose, grape seed extract, bill perry, feverfew, homecoming, soybean, French coastal pine, garlic, ginseng , Tea, ginger, agaritas, mesimacob, purple ipe, active hemicellulose compound (Active— Hemi—Cellulose— Compound: AHCC), yeast betaglecan, maitake, propolis, beer fermentation mother, cereal, plum, chlorella, Barley young leaves, green juice, vitamins, collagen, darcosamine, mulberry leaves, rooibos tea, amino acids, royal jelly, shiitake mycelium extract, spirulina, tannin carrot, taleson, plant fermented food, docosahexaenoic acid (DHA), D Eicosapentaenoic acid (EPA), arachidonic acid (ARA), kelp Cabbage, aloe, Megusurino trees, hops, Chikaraki meat E kissing, Pikujenoru, functional foods sesame, etc. may used in the present invention, 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. 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.

[0208] For example, a ribosome containing DHA can be used as a functional food, nutritional supplement, or health supplement effective for mild senile dementia and memory improvement.

[0209] References such as scientific literature, patents, and patent applications cited herein are incorporated herein by reference in their entirety to the same extent as if each was specifically described. The

[0210] As described above, the power of the present invention exemplified by the preferred embodiment of the present invention. The present invention should not be construed as being limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and literature references cited in this specification should be incorporated by reference as if the contents themselves were specifically described in the present specification. Is understood.

[0211] Hereinafter, the configuration of the present invention will be described in more detail by way of examples, but the present invention is not limited thereto. The reagents used in the following were commercially available unless otherwise specified.

Example

[0212] (Example 1. Preparation of ribosome)

For ribosomes, 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). Prepared. In other words, 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. Then, 46.9 mg of sodium cholate was added, and dissolved in 3 ml 1 of black mouth form Z methanol (1: 1) solution. The solution was evaporated and the precipitate was dried in vacuo to obtain a lipid membrane. The obtained 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 Ν 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).

[0213] (Example 2. Hydrophilization treatment on ribosomal lipid membrane surface)

10 ml of the ribosome solution prepared in Example 1 was subjected to ultrafiltration (fractionated molecular weight: 300,000) using XM300 membrane (Amicon Co., USA) and carbonate buffer (pH 8.5), and the pH of the solution was adjusted. 8.5. Next, 10 mg of the crosslinking reagent bis (sulfosuccinimidyl) suberate (BS ³ ; Pierce Co., USA) was added and stirred at room temperature for 2 hours. Then, to complete the chemical bonding reaction with dipalmitoyl phosphatidylethanolamine § Min and BS ³ on the ribosome film was stirred overnight further under refrigeration. Then, this ribosome solution was subjected to ultrafiltration (fractionated molecular weight: 300,000) with an XM300 membrane and a carbonate buffer (pH 8.5). Next, 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. Is exchanged for N-tris (hydroxymethyl) 3-aminopropanesulfonic acid buffer (pH8.4), and the chemical binding reaction between BS ³ bound to lipid on the ribosome membrane and tris (hydroxymethyl) aminomethane Was completed. As a result, the hydroxyl group of tris (hydroxymethyl) aminomethane was coordinated on the lipid dipalmitoyl phosphatidylethanolamine of the ribosome membrane to make it hydrated and hydrophilic.

[0214] (Example 3. Binding of human serum albumin (HSA) to ribosome membrane surface) The binding of human serum albumin (HSA) to the ribosome membrane surface was performed by a previously reported method (Yamaza ki, N., Kodama, Μ. And Gabius, H. —J. (1994) Methods Enzymol. 242, 56 -65). The coupling reaction method was used. That is, this reaction is a two-step chemical reaction. First, gandarioside present on the surface of 10 ml of the ribosome obtained in Example 2 was converted to 1 ml of N-tris (hydroxymethyl) -3-aminopropanesulfone. 43 mg of sodium metaperiodate dissolved in an acid buffer (pH 8.4) was added and stirred under refrigeration to form periodate. 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. To this ribosome solution, 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

Three

Under stirring, the mixture was agitated and HSA was bound by a coupling reaction between gandioside on the ribosome and HSA. Then, ultrafiltration (fractionated molecular weight: 300,000) was performed to remove free sodium cyanoborate and human serum albumin, and the buffer of this solution was replaced with carbonate buffer (pH 8.5). 10 ml of HSA binding ribosome solution was obtained.

[0215] (Example 4. Preparation of sugar chain)

The sugar chains shown in Table 2B below were used.

[0216] The mass of each sugar chain was measured and pretreated for use in Example 5 below. When using a combination of two or more sugar chains, mix each sugar chain.

[0217] [Table 2]

(Table 2A)

Abbreviation Name (English) Name (Japanese) One manufacturer Product number

SLX Sialyl Lewis X CALBIOCHEM 565950

G4GN N-Acetyllacdosa

N-Acetyllactosamine CALBIOCHEM 345 250 Min

A6 Οί 1 ^_ o Mannobiose α 1 -6 Mannobiose Funakoshi DL-1220-60 [0218] (Table ² B)

(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.

4 3 Add to 0.5 ml aqueous solution and stir at 37 ° C for 3 days, then filter through 0.45 μm filter to complete the amination reaction of the reducing end of the sugar chain. A glycosylamine compound 4 mgZml (aminated sugar chain solution) was obtained. Next, 10 mg of a cross-linking reagent 3, 3, monodithiobis (sulfosuccinimidyl propionate) (DT SSP; Pierce Co., USA) was added to 10 ml of a part of the ribosome solution obtained in Example 3. Stir for 2 hours at room temperature, and then stir under refrigeration, ultrafiltration (fraction molecular weight: 300,000) with XM300 membrane and carbonate buffer (pH 8.5) to remove free DTSSP Thus, ribosome 1 Oml in which DTSSP was bound to HSA on the ribosome was obtained. Next, the glycosamine amine compound (aminated sugar chain solution) is added to the ribosome solution. Solution) 12. 5, 37.5, 125, 250, 500, 1250, 2500 / zl was added, reacted at room temperature for 2 hours, and tris (hydroxymethyl) aminomethane / carbonate buffer (pH 8.5) was added. Thereafter, the mixture was stirred overnight under refrigeration, and the glycosylamine amine compound was bound to DTSSP on ribosome membrane surface-bound human serum albumin. Free sugar chains and tris (hydroxymethyl) aminomethane were removed by ultrafiltration (fraction molecular weight: 300,000) with XM300 membrane and HEPES buffer (pH 7.2). As a result, 10 ml of each ribosome in which the sugar chain shown in Table 2B, human serum albumin and liposome were bound (total lipid amount 26 mg, total protein amount 1.5 mg, average particle diameter lOOnm) was obtained.

(Preparation example 1. Binding of tris (hydroxymethyl) aminomethane onto ribosome membrane-bound human serum albumin (HSA))

In order to prepare a ribosome as a comparative sample, 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. , USA) Add 10mg and stir for 2 hours at room temperature, then refrigerate, and ultrafilter with XM300 membrane and carbonate buffer (pH 8.5) (fraction molecular weight: 300, 000) Free DTSSP was removed to obtain ribosome 10m 1 in which DTSSP bound to HSA on ribosome, and then 26.4 mg of tris (hydroxymethyl) aminomethane (Wako Co., Japan) was added to this ribosome solution. The mixture was stirred for 2 hours at room temperature and then overnight under refrigeration to bind tris (hydroxymethyl) aminomethane to DTSSP on liposomal membrane-bound human serum albumin XM300 membrane and HEPES buffer (pH 7. Ultrafiltration (fraction molecular weight: 300,000) in 2). Since 26.4 mg of tris (hydroxymethyl) aminomethane was present in a large excess, hydrophilic treatment on ribosome membrane-bound human serum albumin (HSA) was completed at the same time. Then, 10ml of ribosome (abbreviation: TRIS) as a comparative sample in which the final product, tris (hydroxymethyl) aminomethane treated with hydrophilicity, human serum albumin, and ribosome were combined (total lipid amount 26mg, total protein amount 1) 5 mg, average particle size lOOnm) was obtained.

(Example 6. Hydrophilization treatment on ribosome membrane surface-bound human serum albumin (HSA)) For the ribosome to which the sugar chain prepared by the means of Example 5 was bound, the ribosome was separately treated on the ribosome by the following procedure. The surface of the HSA protein was treated with hydrophilicity. . Separately, 26.4 mg of tris (hydroxymethyl) aminomethane was added to 10 ml of the sugar chain-modified ribosome, stirred at room temperature for 2 hours, and then overnight under refrigeration, and then XM300 membrane and HEPES buffer (pH 7.2). And ultrafiltered (fraction molecular weight: 300,000) to remove unreacted substances. 0.4 Filtered with a 5 m filter, 10 ml each of the final product, hydrophilic-modified glycosylated liposome complex (total lipid content 26 mg, total protein content 1.5 / zg, average particle size) Diameter lOOnm).

(Example 7. Preparation of ribosome encapsulating fluorescent dye)

(Preparation of cy5.5 labeled human serum albumin solution)

Human serum albumin ZN Tris (hydroxymethyl) 3-aminopropanesulfonic acid buffer (pH8.4) solution (10mgZml), (2ml) in cy5.5ZN Tris (hydroxymethyl) 3 aminopropanesulfonic acid buffer (pH8) 4) 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.

[0222] Ribosomes have been reported (Yamazaki, N., Kodama, M. and Gabius, H. —J.

(1994) Methods Enzymol. 242, 56-65) using a modified cholate dialysis method. In other words, 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. Then, 46.9 mg of sodium cholate was added, and dissolved in 3 ml 1 of black mouth form Z methanol (1: 1) solution. The solution was evaporated and the precipitate was dried in vacuo to obtain a lipid membrane. 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 (ρΗ8.4) (fractional molecular weight: 10 000) to obtain uniform fluorescence 10 ml of a ribosome particle suspension containing the dye was prepared. [0223] 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 8. Treatment of hydrophilic dig on ribosomal lipid membrane surface encapsulating fluorescent dye)

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晚攪拌lipid on the ribosome film dipalmitoylphosphatidyl E Tano Ruamin and BS ^3. Then, this ribosome solution was subjected to ultrafiltration (fractionated molecular weight: 300,000) with an XM300 membrane and a carbonate buffer (pH 8.5). Next, 40 mg of tris (hydroxymethyl) aminomethane dissolved in 1 ml of carbonate buffer (pH 8.5) was added to 10 ml of liposome solution. Next, 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. Was replaced with N-tris (hydroxymethyl) 3-aminopropane sulfonate buffer (pH 8.4) to complete the chemical binding reaction between BS ³ bound to lipid on the ribosome membrane and tris (hydroxymethyl) aminomethane. As a result, the hydroxyl group of tris (hydroxymethyl) aminomethane was coordinated on the lipid dipalmitoylphosphatidylethanolamine of the ribosome membrane to make it hydrated and hydrophilic.

[Example 9: Binding of human serum albumin (HSA) to the surface of a ribosome membrane encapsulating a fluorescent dye)

The binding of human serum albumin (HSA) to the ribosome membrane surface was performed by a previously reported method (Yamaza ki, N., Kodama, Μ. And Gabius, H. —J. (1994) Methods Enzymol. 242, 56 -65). The coupling reaction method was used. That is, this reaction is a two-step chemical reaction. First, gandarioside present on the surface of 10 ml of ribosome obtained in Example 2 is converted to 1 ml of N tris (hydroxymethyl) -3-aminopropanesulfonic acid. Add 10.8 mg of sodium metaperiodate dissolved in buffer solution (pH 8.4) and stir at 7 ° C. Stir to periodate. 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. To this ribosome solution, 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

Three

(Example 10. Preparation of sugar chain)

A sugar chain was prepared by the same procedure as in Example 4.

(Example 11. Binding of sugar chain onto ribosome membrane surface-bound human serum albumin (HSA) encapsulating fluorescent dye and hydrophilization of linker protein (HSA))

2 mg of each sugar chain prepared in Example 10 was dissolved in purified water, and 0.25 g NH 4 HCO

4 3 was added to a 0.5 ml aqueous solution and stirred at 37 ° C for 3 days, and then filtered through a 0.45 m filter to complete the amination reaction at the reducing end of the sugar chain. Of 4 mgZml (aminated sugar chain solution) was obtained. Next, 10 mg of a cross-linking reagent 3, 3, dithiobis (sulfosuccinimidyl propionate) (DTSSP; Pierce Co., USA) was added to 10 ml of a portion of the ribosome solution obtained in Example 9. Stir for 2 hours at room temperature, then under refrigeration, ultrafilter with XM300 membrane and carbonate buffer (pH 8.5) (molecular weight cut off: 300,000) to remove free DTSSP. Ribosome 1 Oml in which DTSSP was bound to HSA on the ribosome was obtained. Next, add the above glycosamine amine compound (aminated sugar chain solution) 12.5, 37.5, 125, 250, 500, 1250, 2500 / zl to this ribosome solution, and add 2 at room temperature. React for hours, add tris (hydroxymethyl) aminomethane / carbonate buffer (pH 8.5), then stir overnight under refrigeration, and add glycosylamine to DTSSP on ribosomal membrane-bound human serum albumin. Bonding of the compounds was performed. Ultrafiltration (fraction molecular weight: 300,000) with XM300 membrane and HEPES buffer (pH 7.2), free sugar chain and tris (hydroxymethyl) Minomethane was removed. As a result, 10 ml of each ribosome in which the sugar chain shown in Table 2B, human serum albumin, and liposome were bound (total lipid amount 38 mg, total protein amount 15 mg, average particle size 1 OOnm) was obtained.

[0228] 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)) [0229] Regarding the ribosome to which the sugar chain prepared by the means of Example 11 was bound, the following procedure was performed on the ribosome. The surface of the HSA protein was subjected to hydrophilic treatment. Separately, 26.4 mg of tris (hydroxymethyl) aminomethane was added to 10 ml of the glycosylated liposomes, stirred at room temperature for 2 hours and then overnight under refrigeration, and then XM300 membrane and HEPES buffer (pH 7.2). ) And ultrafiltered (fraction molecular weight: 300,000) to remove unreacted substances. Filter through a 45 μm filter to obtain 10 ml each of the hydrophilized glycosylated ribosome complex (total lipid content 38 mg, total protein content 15 mg, average particle size 1 OOnm). It was.

(Example 13. In vivo imaging in tumor-bearing mice)

(Production of tumor-bearing mice)

Ehrlich ascite subcutaneously in the right thigh of a mouse (ddyY, male, 7 weeks old)

Tumor (EAT) cells were transplanted 5 × 10 ⁶ cells and used for experiments 7-10 days later.

[0231] (Evaluation method)

1/10 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 μg of lipid) was administered, and image data immediately after administration were collected.

[0232] As a control, the same amount of ribosome particles was administered with sugar chains attached! /. Image data was collected over time. All image data were taken from the ventral side.

[0233] (Result)

As shown in Figure 1, there is no difference between the presence and absence of sugar chains immediately after ribosome administration! / After 1 day, sugar chain-modified ribosome (Kl) accumulated more strongly at the tumor site than without sugar chain. Two days later, it was confirmed that the sugar chain-modified ribosome (K1) was significantly accumulated at the tumor site. From this result, it was found that the tumor site can be imaged by K1-3 ribosome. When the ribosome dose was changed from 200 μ1 to 50 μ1 and 1Z4, the knockdown decreased and it was confirmed that sugar chain-binding liposomes were accumulated at the tumor site even after 8 hours. One day later, there was a clear difference from the absence of sugar chain at the tumor site. From this result, it was proved that imaging of the tumor site with K1-3 ribosome was possible in a short time and in a short time (Fig. 2). In the case of a ribosome dose of 200 1, it was confirmed that the accumulation of sugar chain-modified ribosome (K1) was maximized 2 days after administration, and then the signal gradually weakened. On the other hand, ribosomes without sugar chains were powerful without significant changes. From this result, it was found that K1-3 ribosomes are metabolized to the tumor site and can be seen in real time by imaging (Fig. 3).

[0234] As shown in Fig. 4, the sugar chain-modified ribosome (Κ3) 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 Κ3-3 ribosome, is optimal for imaging the tumor site, and that the sugar chain is specific.

(Example 14. Experiment using rheumatoid arthritis model mouse)

(Production of human rheumatoid arthritis model mice)

Mice (BalbZc, female, 8 weeks old) were administered with 200 l (2 mg) of monoclonal antibody (Chondrex) for inducing arthritis in the tail vein. Two to five days after administration, LPS (Lipopolysaccharide) 100 1 (50 1) was intraperitoneally administered to mice. Mice developed arthritis 3-4 days after administration.

[0236] (Evaluation method)

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.

[0237] (Result)

As shown in FIG. 5, it was confirmed that 1 day after administration of the sugar chain-modified ribosome (K1), it was accumulated in the inflamed site. Then, after 2 days and 3 days, the signal gradually weakened. From this result, it was found that imaging of the inflammatory site by K13 ribosome is possible. In addition, the ability of K1-3 ribosomes accumulated in the inflamed site to be metabolized was visualized in real time by imaging. As shown in FIG. 6, it was confirmed that 1 day after administration of the sugar chain-modified liposome (K1), it was accumulated in the inflamed site. After that, as in Fig. 5, the signal began to weaken after 2 days. This result showed reproducibility of imaging and specificity of the inflammatory site by K1-3 ribosome.

[0238] As shown in Fig. 7, when sugar chain-modified ribosome (K1) binds to the surface of the ribosome and changes the density of sugar chains, the amount of accumulation at the inflammatory site is compared. Was. K1-4 was also not as prominent as K1 3, but accumulated at the site of inflammation. Increasing the density of Kl-5 and Kl-6 weakened the signal at the inflammatory site more than ribosomes without sugar chains. From these results, it is suggested that for inflammatory site imaging by glycan-modified ribosomes (K1), the density of glycans bound to the ribosome surface such as K1-3 and K1-4 is better. I understood. Moreover, the specificity by the modified bond density of the sugar chain on the ribosome surface was shown. Glycan-modified ribosomes (K3) did not show any difference from ribosomes with no sugar chain after one day. Based on the results shown in Fig. 7, there is a difference in the accumulation of ribosomes at the inflammatory site between the sugar chain-modified ribosome (K1) and the sugar chain-modified ribosome (K3). It was shown to accumulate (Figure 8).

[0239] In the sugar chain-modified ribosome (K2), it was confirmed that K2-3 was accumulated at the site of inflammation after 1 day. From this result, it was proved that it is possible to image the inflammatory site with K2-3 ribosome. In addition, based on the results shown in FIGS. 7 to 9, the specificity of the sugar chain is shown by the degree of accumulation in the inflamed site, and in the imaging of the inflamed site, the density of the sugar chain bound to the ribosome surface is low. This is better (Fig. 9). Sugar chain K1 showed a significant difference between sugar chain level 3 (K 1-3) and sugar chain level 4 (K1 -4). Accumulated in normal mice Yes. These results indicate that 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). For sugar chain K1, 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)

[0240] Sugar chain K3 did not accumulate in the inflamed site even when the sugar chain density was changed. Based on these results and Figs. 8 and 12, the sugar chain-modified ribosome (K3) does not accumulate at the inflammatory site even if the density of the sugar chain bound to the ribosome surface is changed, and binds to the ribosome surface. Specificity of the glycemic chain to the inflamed site was particularly strong (Fig. 13).

[0241] (Example 15. Experiment using normal 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.

[0242] (Evaluation method)

A 1/10 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.

[0243] (Result)

Figure 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.

[0245] 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.

[0246] 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.

[0247] 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.

[0248] FIG. 19 shows spleen data. The signal is weak overall, but partially (K2—3, Κ2

6) and other areas with high signal. From this result, it was found that the specificity of accumulation in the spleen due to the type of sugar chain and the difference in the modified bond density can be seen as imaging.

[0249] FIG. 20 shows cardiac data. When the sugar chain density was changed, a difference in signal intensity was observed. 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.

[0250] 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.

[0251] FIG. 23 shows whole body scan data of Κ2-3 (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.

[0252] 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. From this result, it was clear that the ribosome was metabolized and excreted into the liver and bladder by imaging. 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.

(Example 16. Quantitative analysis of ribosome)

(I. Protein quantification) The amount of HSA encapsulated in the ribosome and the total tank mass of HSA coupled to the ribosome surface were measured by the BCA method.

[0255] Micro BCA ™ 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.

[0256] As a standard solution, 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. Reagents A, B, and C attached to the kit were mixed so that reagent A: reagent B: reagent C = 48: 2: 50, and 150 1 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.

[0257] [Table 3]

[0258] (II. Lipid determination)

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

Cholesterol was used.

[0259] As a standard solution, dilute standard substance (50mgZml: cholesterol) with PBS buffer solution, 0, 0.1, 0.25, 0.5, 0.75, 1, 5, ₈ Ζ20 / ζ 1 solution Was prepared. Cy5.5-encapsulated sugar chain-modified ribosome was diluted 5-fold with PBS buffer to prepare a sample solution. The standard solution and the sample solution were each dispensed into a test tube for 20 minutes. To each test tube, Triton X-100 (10% solution) 17 1 was added and stirred, and then allowed to stand at 37 ° C. for 40 minutes. 3001 enzyme reagent from Detamina TC555 kit was added and stirred, and then allowed to stand at 37 ° C for 20 minutes. Absorbance was measured at 540 nm, a calibration curve was prepared with a standard solution, the amount of cholesterol in the liposome was measured, and the amount of lipid was determined.

Cholesterol power Conversion formula for lipid content

Lipid content Ζ ⁵ 〇 / Ζ 1) = Cholesterol content gZSO / zl) X 4.51 (Conversion factor) The quantitative results of lipid content are shown below.

[0260] [Table 4]

(Table 4 Quantitative results of lipid content) cy5.5 Encapsulated sugar

Chain-modified liposome

(I / 50ju l) cy5.5 K1-0 191

cy5.5 K1-1 168

cy5.5 K1-2 178

cy5.5 K1-3 187

cy5.5 K1-4 199.8 cy5.5 K1-5 166.2 cy5.5 K1-6 161.3 cy5.5 K2-0 214.4 cy5.5 K2-1 215.3 cy5.5 K2-2 183.5 cy5.5 K2-3 217.2 cy5 .5 K2-4 221

cy5.5 K2-5 198.7 cy5.5 K2-6 205.3 cy5.5 K3-0 245.6 cy5.5 K3-1 212.7 cy5.5 K3-2 200.1 cy5.5 K3-3 198.6 cy5.5 K3-4 204.2 cy5 .5 K3-5 197.7 cy5.5 K3-6 208.6 [0261] [Table 5]

(Table 5 Ratio of protein to lipid)

[0262] (III. Particle size distribution and particle size)

Ribosome particles were diluted 50-fold with purified water and measured using a Zetasizer Nano (Nan-ZS: MA LVERN Co. LTD).

[0263] [Table 6] Measured particle size (nm) result

(Example 17. Preparation of fluorescent-labeled substance-attached ribosome I)

(Preparation of cy5.5 labeled human serum albumin solution)

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. [0265] The gandarioside present on the 10 ml ribosome membrane surface obtained in Example 2 was dissolved in lml N-tris (hydroxymethyl) -3-amaminopropanesulfonate buffer (pH 8.4). Add 43 mg of sodium metaperiodate and stir overnight under refrigeration for periodate. Free filtration of sodium periodate was removed by ultrafiltration (fraction molecular weight: 300, 000) with XM300 membrane and PBS buffer (pH 8.0). N-Tris (hydroxymethyl) -3- Exchange minopropane sulfonate buffer with PBS buffer (pH 8.0) to obtain 10 ml of oxidized liposomes. To this ribosome solution, add 20 mg of cy5.5-labeled human serum albumin solution and react at room temperature for 2 hours, then 2M NaBH CNZPBS buffer (pH 8.0)

Three

Add 1001 and stir for 2 hours at room temperature and overnight under refrigeration to allow cy5.5-labeled HSA to bind by coupling reaction between gandarioside on ribosome and cy5.5-labeled HSA. Next, ultrafiltration (fractional molecular weight: 300,000) was performed to remove free sodium cyanoborate and human serum albumin, and the buffer of this solution was replaced with carbonate buffer (pH 8.5). Obtain 10 ml of cy5.5-labeled HSA-binding ribosome solution.

[0266] (Preparation of sugar chain)

A sugar chain is prepared in the same manner as in Example 4.

[0267] (Ribosomal membrane surface binding cy5.5 Binding of sugar chains onto 5-labeled human serum albumin (HSA)) 2 mg of each sugar chain prepared in Example 4 was dissolved in purified water, and 0.25 g of NH HCO was dissolved.

4 3 Add to 0.5 ml aqueous solution and stir at 37 ° C for 3 days, then filter through 0.45 μm filter to complete the amination reaction of the reducing end of the sugar chain. Glycosamine amine compound 4mgZml (aminated sugar chain solution) is obtained. Next, 10 mg of a crosslinking reagent 3, 3, monodithiobis (sulfosuccinimidyl propionate) (DTSS P; Pierce Co., USA) was added to 10 ml of a part of the ribosome solution obtained in Example 3 at room temperature. For 2 hours, followed by agitation under refrigeration, ultrafiltration (fraction molecular weight: 300, 000) with XM300 membrane and carbonate buffer (pH 8.5) to remove free DTSSP, DTSSP Obtain 10 ml of liposomes bound to cy5.5-labeled HSA on the ribosome. Next, transfer the above glycosylamine compound (amino saccharose solution) 12.5, 37.5, 125, 250, 500, 1250, 2500 1 to this ribosome solution and react at room temperature for 2 hours. Add tris (hydroxymethyl) aminomethane Z carbonate buffer (PH 8.5), and then stir overnight under refrigeration to bind to ribosome membrane surface cy5.5. 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.

[0268] (Ribosomal membrane surface-bound cy5.5-labeled human serum albumin (HSA) hydrophilization treatment) For the ribosomes to which the above sugar chains are bound, the cy5.5-label on the liposomes is separately treated by the following procedure. Perform hydrophilic treatment of the HSA protein surface. Separately, 26.4 mg of tris (hydroxymethyl) aminomethane was added to 10 ml of the glycosylated liposome, stirred at room temperature for 2 hours, and then overnight under refrigeration, and then XM300 membrane and PBS buffer (pH 7. Perform ultrafiltration (fraction molecular weight: 300, 000) in 2) to remove unreacted substances. Filter through a 0.45 m filter to obtain 10 ml each of the glycosylated ribosome complex that has been hydrophilized as the final product.

(Example 18. Preparation of fluorescent-labeled substance-attached ribosome II)

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.

[0270] 10 ml of the sugar chain-modified ribosome obtained in Example 6 was ultrafiltered (fraction molecular weight: 300,000) with XM300 membrane and PBS buffer (pH 7.2), and cy3 or cy5Z dimethylformamide (D MF ) (pH 8. ⁴⁾ solution ⁽² 0mgZml) by mixing 100 1, after stirring for ² hours at room temperature and stirred overnight under refrigeration. Free fluorescent dye is removed by ultrafiltration (fraction molecular weight: 300,000) with XM300 membrane and HEPES buffer (pH 7.2). The final product, fluorescently labeled substance-attached liposome, is obtained.

(Example 19. Imaging using a ribosome encapsulating a fluorescent dye)

(Preparation of Cy5.5 encapsulated sugar chain-modified ribosome (K1))

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.

Sci. 1989, 41, 249—267, Yamazaki, N., Komada, M., Gabius, HJ. Methods. Enzymol. 1994, 242, 56—65 Prepared. [0272] Dipalmitoylphosphatidylcholine (16.8 mg), cholesterol (10. lmg), dicetyl phosphate (1.8 mg), ganglioside (14.6 mg), dipalmitoylphosphatidylethanolamine (2.3 mg) and Sodium cholate (46.9 mg) was mixed. This mixture was dissolved in 3 ml of Kloform-form Z methanol (1: 1, vZv) solution. The solvent was evaporated using a rotary evaporator at 30 ° C. and dried under reduced pressure to obtain a fat film. The lipid film was dissolved in 3 ml of tris (hydroxymethyl) methylaminobutane sulfonic acid buffer (TAPS, pH 8.4) and, after sonication, a micelle suspension was obtained.

[0273] 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 (ρΗ8.4) 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.

[0274] A solution of HSA with Cy5.5 (3 ml) was mixed with the micelle suspension described above. This mixed solution was subjected to ultrafiltration with TAPS (ρ CO8.4) by PM10 (Amicon CO., LTD) to remove the remaining HSA having Cy5.5 to obtain a ribosome solution (10 ml). 10mg bis (sulfosuccinimidyl) suberate (BS; PIERCE

Three

CO., LTD) (crosslinking agent) was added to the ribosome solution (10 ml), and the mixture was stirred at 20 to 25 ° C. for 2 hours and further stirred at 4 ° C. overnight. BS was bound to the ribosome surface. Next

Three

Add 40 mg of tris (hydroxymethyl) aminomethane, stir at 20-25 ° C for 2 hours, and then stir overnight at 4 ° C to bind tris (hydroxymethyl) aminomethane to BS.

3 This was ultrafiltered with XM300 (Amicon CO., LTD) to remove the remaining tris (hydroxymethyl) aminomethane. A coupling method was used to bind HSA to the ribosome surface. To acidify the ribosome surface, 10.8 mg of sodium periodate was added to the ribosome solution (10 ml) and stirred at 4 ° C. This was ultrafiltered with XM300 (Amicon CO., LTD) to remove the remaining sodium periodate. 2 Omg of HSA was added thereto and stirred at 20-25 ° C for 2 hours. Then 3. Sodium borohydride was added and stirred at 20 to 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 sodium cyanoborohydride.

[0275] The sugar chain was bound to the ribosome surface via 3,3 dithiobis (sulfosuccinimidylpropionate) (DTSSP; PIER CE CO., LTD). 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

The mixture was stirred at ° C for 3 days. Amino 匕 SLX, 25

1 (density 02: 1 ^ 1—3), 100, ug / ml (¾ ¾D3: Kl -4), 200, ug / ml (¾ ¾D4: Kl 5) and 500 μg / ml (density D5: Kl— The final concentration of 6) was reached, and the mixture was stirred at 20-25 ° C for 2 hours.

[0276] Tris (hydroxymethyl) aminomethane was calored until a final concentration of 132 mg / ml was reached and stirred at 4 ° C to repeatedly hydrophilicize the ribosome surface. This solution was ultrafiltered through XM 300 (Amicon CO., LTD) to remove residual SLX and tris (hydroxymethyl) aminomethane.

[0277] (Preparation of ribosome without sugar chain)

Preparation of ribosomes without sugar chains was performed in the same manner as in the case of sugar chain-modified liposomes, except for the process of attaching sugar chains. The binding of N-acetyllactosamine (G4GN: Calbiochem CO., LTD) to the ribosome surface was also performed in the same manner as in the case of the sugar chain-modified ribosome.

[0278] (Quantitative analysis of ribosomes)

(Measurement of lipid content of ribosome)

Using the Deter miner 555 kit (1 ^^^ 8 CO., LTD), the lipid content of the resulting sugar chain-modified ribosome (K1) and sugar chain-free ribosome was 0.5%

Measured as total cholesterol in the presence of TritonX-100. Then the fatty acid The total amount was calculated from the molar ratio of each lipid. Protein content is described in Yamazaki, NJ Membrance Sci. 1989, 41, 249—267, Yamazaki, N., Komada, M., Gabius, H-J. Methods. Enzymol. 1994, 242, 56—65 As described above, measurement was performed using Micro BC A ™ Protein Assay Reagent (PIERCE CO., LTD) in the presence of 1% sodium dodecyl sulfate (SDS).

[0279] The lipid content and protein content of the sugar chain-modified ribosome (K1) were 3.7 mg / ml and 1.2 mgZml, respectively. For sugarless ribosomes, a lipid content of 3.8 mgZml and a protein content of lmgZml were obtained.

[0280] (Measurement of ribosome particle size and zeta potential)

The particle size and ζ potential were measured at 25 ° C. using Zetasizer Nano—S 90 (Malvern CO., LTD). This ribosome solution was diluted 50 times with distilled water for measurement. The particle size of both sugar-modified ribosome (K1) and sugar-free ribosome showed almost uniform distribution. The average particle size was about lOOnm (Figure 41). The ζ potential indicating the charge on the surface of the liposome membrane was negatively charged and was −40 mV for both ribosomes. After storage for 6 months at 4 ° C, the particle size distribution was almost the same as that immediately after preparation, indicating the stable properties of these ribosomes (Figure 42).

(Example 20. Experiment using human rheumatoid arthritis model mouse)

(Preparation of human rheumatoid arthritis model mice and administration of ribosomes)

A human rheumatoid arthritis model mouse was prepared by the following method. For induction of human rheumatoid arthritis, 200 / z l ^ mg) monoclonal antibody (Chondrex CO., LTD) was administered from the tail vein force of BalbZc mice (female, 8 weeks old). Four days later, 100 1 (50 g) lipopolysaccharide (LPS) was administered intraperitoneally. The mice were used for experiments after 3-4 days. A 150 1 Nembutal solution (Dainippon Pharmacoeutical CO., LTD) diluted 10-fold with physiological saline was administered intraperitoneally, and the model mice were anesthetized. Subsequently, sugar chain-modified ribosomes (K1) or glycosomes without sugar chains were also administered to the tail vein (50 1 per mouse).

[0282] (Example 21. Imaging using explore Optix)

Using eXplore Optix (GE Healthcare CO., LTD), Cy5.5 fluorescent signal Nulls were monitored before the injection, at 0 and 24 hours after injection, in the inflammatory area of the same mouse (back of the hind limb) (excitation: 680 nm, emission: 700 nm).

[0283] Comparing the accumulation of sugar chain-modified ribosomes (K1) and sugar chain-free liposomes in the inflamed area 24 hours after injection, sugar chain-modified ribosomes (K1) accumulate more than sugar-free ribosomes. (Fig. 43).

[0284] (Confirmation of sugar chain specificity)

In order to confirm that this accumulation is SLX specific, the sugar chain modified ribosome (K2) with G4GN bound to the ribosome surface was used in the same manner as the sugar chain modified ribosome (K1) of this example. Prepare with D2 sugar chain density (K2-3). Glycosylated ribosomes (K1 and K2) were also administered with tail vein force (501Z mice) and ribosome accumulation was examined 24 hours after injection. As shown in FIG. 44, the accumulation of sugar chain-modified ribosome (K2) was almost the same level as that of ribosome without sugar chain, and only the sugar chain-modified ribosome (K1) was specifically accumulated in the force-inflammatory region.

[0285] (Effect of sugar chain density on specific accumulation in the inflamed area)

In order to clarify the relationship between the specific accumulation of sugar chain-modified ribosomes (K1) in the inflammatory region and the sugar chain density on the surface, sugar chain-modified ribosomes with various sugar chains (Kl ) (Dl ~ 5) was administered (50 1Z mice), and the degree of ribosome accumulation was compared (Fig. 45).

) o

[0286] Interestingly, particularly when the sugar chain density was D2, it showed the highest accumulation in the inflammatory region. In the sugar-modified ribosome (K1), the accumulation in the inflamed area decreased when the sugar chain density was higher than D2. It was found that the sugar chain density on the surface of the ribosome has a significant effect on accumulation. It became clear that there was an optimal density of sugar chains for efficient accumulation in the inflamed area. Therefore, the sugar chain-modified ribosome (density D2: K1-3) is a remarkably effective type of sugar chain for efficiently accumulating in the inflammatory region and has the optimal binding density! / ヽYeah.

(Example 22. Imaging with a scanning microscope)

(Olympus IV100 observation)

(Method) Scanning fluorescence microscope IV confirms that the force accumulated in the inflamed area is in the blood vessel, or that the vascular force is also transferred to the surrounding tissue. — 100 (OLYMPUS., Co. LTD). Observation was performed with an ultrafine stick objective lens (IV—OB35F22W20) having a diameter of IV-100 of 1.3 mm close to the observation site. Anesthetized with 1/10 Nembutal solution administered intraperitoneally into arthritic mice and administered cy5.5-encapsulated glycosome-modified ribosome (K1-3) (100 1: lipid amount 380 g) from the tail vein. did. As a control, the same amount of ribosome not bound to a sugar chain was administered. The image data was collected over time. All image data were observed on the back of the hind legs.

[0288] In addition, atalidine orange (150 μ 1: 0.5%, w / v%) was administered from the tail vein of mice just before observation to stain leukocytes in blood vessels. At 488 nm and Em 526 nm, fluorescence was observed at ataridine range (green), and Ex 633 nm and Em 693 nm at Cy5.5 (orange).

[0289] (Result)

It was confirmed whether the K1 3 ribosome accumulated in the inflammatory site stayed in the blood vessel, or whether the vascular force was also transferred to surrounding tissues. As shown in Fig. 46, at 10 and 30 minutes after administration, no attachment or aggregation of ribosomes to the blood vessel wall was observed, and ribosomes flowing in the blood vessels were confirmed. At 6 hours after administration, only K13 ribosome was observed to adhere partially to the blood vessel wall. On the other hand, such attachment was not confirmed in ribosomes without sugar chains. At 24 hours after administration, K1-3 ribosomes migrated to the tissues around the blood vessels, and the adhesion along the blood vessels observed 6 hours after the inner wall of the blood vessels was strong. After 48 h, the accumulation of K1-3 ribosomes in the perivascular tissues increased more than after 24 h.

[0290] (Summary)

Previously, the LPS-induced E-selectin expression pattern on human umbilical vein (HUVEC) has been reported to form partial and dense aggregates on non-uniform cells (Joseph. K. Welply., S. Zaheer Abbas., Peter Scudder. Glycob iology. 1994, 4, 259—265, Muligan, MS, Polley, MJ, Bayer, RJ J. Clin. Invest. 1992, 90 (4), 1600 —1607, Pober, JS, Bevilacqua, D.L., Mendrick, LA, Lapierre, LAJ Immunol. 1986, 136 (5), 1680—1 687). In this example, it was confirmed that the sugar chain-modified ribosome (K1-3) liposome existing in the blood vessel rolls from the blood vessel to the blood vessel inner wall and migrates to the tissue around the blood vessel. This indicates that the glycosylated ribosome (K1) force recognized E-selectin expressed heterogeneously on the vascular endothelium and migrated with partial binding.

[0291] (Example 23. Experiment using tumor-bearing mice)

(Creation of tumor-bearing mouse model and administration of ribosome)

Accumulation of sugar chain-modified ribosome (K1) and sugar chain-free ribosome in the tumor region was tested using tumor-bearing mice prepared by the following method. EAT cells (5 × 10 ⁶ cell Z mice) were transplanted into the right femoral region of ddY mice (male, 7 weeks old) and the mice were used for experiments 7-10 days later. For anesthesia, 300 1 Nembutal solution diluted 10-fold with physiological saline was administered intraperitoneally. Subsequently, sugar chain-modified ribosome (K1) or sugar chain-free ribosome was administered from the tail vein (200 μ1Z mice).

[0292] (Example 24. Imaging with explore Optix)

Using explore Optix (excitation: 680 nm, emission: 700 nm), the tumor area (back of the hind limb) of the same mouse was examined before administration, at 0, 24, 48, 72 and 72 hours after administration. And 96 hours later. As shown in FIG. 47, the sugar chain-modified ribosome (K1) was significantly accumulated in the tumor region. This accumulation increased gradually up to 48 hours. On the other hand, the accumulation of ribosomes without sugar chains was ζ ·, which was lower than that of sugar chain-modified ribosomes (K1) after 96 hours.

[0293] The whole body was scanned 96 hours after injection to examine the distribution of fluorescence in the body. Strong fluorescence signals were detected in the liver, bladder and tumor areas (see Figure 48). ₀ These signals were analyzed by fluorescence lifetime. Only Cy5.5 (life, 1.8 ns) bound to HSA was detected in the tumor area. However, in the liver and bladder, both free Cy5.5 (lifetime, 1.5 ns) and HSA-bound Cy5.5 (lifetime, 1.8 ns) were identified.

[Example 25. Imaging with a scanning microscope]

(Olympus IV100 observation)

(Method) 2 × 10 ⁶ cells of Ehrlich ascite tumor (EAT) cells were transplanted subcutaneously into the right thigh of a mouse (BalbZc, female, 8 weeks old) and used for experiments 7-10 days later.

[0295] Scanning fluorescent microscope IV-100 (OLYM PUS.) Confirms whether the K1-3 ribosome accumulated in the tumor-bearing site remains in the blood vessel, or the vascular force is also transferred to surrounding tissues. , Co. LTD). Observation was performed with an IV-100 ultrafine stick objective lens (IV-OB35F22W20) with a diameter of 1.3 mm close to the observation site. An lZlO nebutal solution was administered intraperitoneally into the peritoneal cavity of mice bearing cancer and anesthetized. Using fluorescence imaging device IV-100 (OLYMPUS), image data of blood vessels and surrounding tissues of cancer-bearing sites were collected. From the tail vein, cy5.5-encapsulated sugar chain-modified ribosome (K1-3) (100 1: lipid amount 380 g) was administered. As a control, the same amount of liposomes to which no sugar chain was bound was administered. 48 hours after administration, blood vessels and surrounding tissues at the cancer site (right thigh) were observed using IV-100 (OLYMPUS). Immediately before the observation, atalidine orange (150 ^ 1: 0.5%, wZv%) was administered from the tail vein of the mouse to stain leukocytes in the blood vessel. Fluorescence of Atalidine Orange (green) was observed at Ex 488 nm and Em 526 nm, and Cy5.5 (orange) was observed at Ex 633 nm and Em 693 nm.

[0296] (Result)

We confirmed whether the K1 3 ribosome accumulated in the tumor-bearing site stayed in the blood vessel or moved to the tissue around the vascular force. As shown in FIG. 49, the K1-3 liposome migrated from the blood vessel to the extravascular tissue 48 hours after administration, and was accumulated in the cells of the extravascular tissue. In the case of ribosomes without sugar chains, the amount of accumulation was less than that of K1-3 ribosomes at 48 hours after administration, but fluorescence was confirmed in extravascular tissues. This was thought to be due to the passive transition of the vascular endothelium around the cancer to the tissue even in the case of liposomes without sugar chains.

[0297] (Summary)

It has been reported that retention of ribosomes in blood vessels has a significant impact on accumulation in inflammatory and tumor areas, and this accumulation is increased by prolonging retention in blood vessels (Drummond, DC, Meyer, O., Hong, Κ., Kirpotin, D. Β. Pharmac ological. Rev. 1999, 51, 691—743, Kawakita, Y., Yamazaki, N., Kojim a, S. Radioisotopes. 2000, 49, 339—345, Yamazaki, N. US patent. 200 3—0143267).

[0298] As measures to prolong retention of ribosomes in blood vessels, to date, "avoidance of uptake of reticuloendothelial system (RES) into liver and spleen", "avoidance of macrophage engulfment" and "vascular endothelium" Avoidance of non-specific adsorption by cells such as cells and leukocytes ”(Drummond, DC, Meyer, O., Hong, Κ., Kirpotin, D. Β. Pharmacological. Rev. 1999, 51, 691—743).

[0299] Sugar-modified ribosomes (Kl) and cells such as vascular endothelial cells, erythrocytes and leukocytes are negatively charged. From this, the sugar chain-modified ribosome (K1) and these cells repulse each other electrically. Therefore, the sugar chain-modified ribosome (K1) is not adsorbed nonspecifically to such cells. Furthermore, by making the ribosome surface hydrophilic, adsorption of opsonin protein in plasma and phagocytosis by macrophages can be avoided, thus extending retention of ribosomes in the bloodstream.

[0300] As a result, the sugar chain-modified ribosome (K1) exhibits an affinity for E-selectin and excellent retention in the blood vessel, and is thought to accumulate specifically and efficiently in the inflammatory region. Therefore, sugar chain-modified ribosome (K1) is useful as an in vivo fluorescent imaging reagent. By selecting the appropriate sugar chain and density, liposomes with sugar chains can be used to transfer various substances (fluorescent substances, chemicals, proteins, genes, etc.) to a specific desired region (disease). Including the region). This indicates that these types of liposomal dynamic targeting DDS can be useful tools.

[0301] (Example 26. Imaging using fluorescent-labeled substance-attached ribosome)

(Preparation of fluorescent labeling substance external ribosome)

Using SLX as a sugar chain, a fluorescently labeled external liposome is prepared using the same method as in Example 17.

[0302] (Preparation of ribosome without sugar chain)

Preparation of ribosomes without sugar chains is prepared using a method similar to Example 19.

[0303] (Quantitative analysis of ribosome)

(Measurement of lipid content of ribosome) The lipid content of the obtained sugar chain-modified ribosome (K1) and sugar chain-free ribosome is measured using the same method as in Example 19. The total amount of fatty acids is then calculated from the molar ratio of each lipid. The protein content is measured in the presence of 1% sodium dodecyl sulfate (SDS).

[0304] (Measurement of ribosome particle size and zeta potential)

Using the same method as in Example 19, the particle size and ζ potential are measured. The particle size of both sugar-modified ribosomes (K1) and non-glycosylated ribosomes shows an almost uniform distribution. The zeta potential, which indicates the charge on the ribosome membrane surface, is negatively charged and is -40 mV for both liposomes. After storage for 6 months at 4 ° C, the particle size distribution is almost the same as that immediately after preparation, indicating the stable properties of these ribosomes.

[Example 27. Experiment using human rheumatoid arthritis model mouse]

A human rheumatoid arthritis model is prepared in the same manner as in Example 20. For anesthesia, a 150 μl Nembutal solution (Dainippon Pharaceutical CO., LTD) diluted 10-fold with saline is administered intraperitoneally. Subsequently, sugar chain-modified ribosomes (K1) or sugar chain-free ribosomes are also administered by tail vein force (50 1 per mouse).

(Example 28. Imaging using explore Optix)

Using the same method as in Example 21, the fluorescence signal of Cy5.5 is monitored in the inflammatory area of the same mouse (back of the hind limb) before injection, at 0 and 24 hours after injection (excitation: 680 nm, emission: 700 nm).

[0307] Comparing the accumulation of glycosylated ribosomes (K1) and glycosylated liposomes in the inflamed area 24 hours after injection, glycosylated ribosomes (K1) accumulate more than glycosylated ribosomes To be confirmed.

[0308] (Confirmation of sugar chain specificity)

In order to confirm that this accumulation is SLX-specific, the sugar chain-modified ribosome (K2) in which G4GN is bound to the ribosome surface was converted to the sugar chain density of D2 (K 2-3) by the same method as in Example 21. Prepare with. These ribosomes (K1 and K2) are administered via the tail vein (50 1 per mouse) and ribosome accumulation is examined 24 hours after injection. Glycomodified ribosome Accumulation of (K2) is almost the same level as in the case of ribosome without sugar chain, and it is confirmed that only the sugar chain-modified liposome (K1) accumulates specifically in the inflammatory region.

[0309] (Effect of sugar chain density on specific accumulation in the inflamed area)

In order to clarify the relationship between the specific accumulation of sugar chain-modified ribosomes (K1) in the inflammatory region and the sugar chain density on the surface, sugar chain-modified ribosomes with various sugar chains (Kl : D1-5) (50 1 per mouse) and compare the degree of ribosome accumulation

[0310] As a result, sugar chain-modified ribosome D2 (K1-3) shows the highest level of accumulation. This accumulation is lower for sugar chains with a higher density than for D2 (K1-3). In addition, it is confirmed that sugar chain-modified ribosome D2 (K1-3) has higher accumulation than D1 (K1-2). From the above, there is an optimal sugar chain density for accumulation in inflammation!

[0311] (Example 29. Imaging with a scanning microscope)

Using the same method as in Example 22, confirm whether K1-3 ribosomes accumulated in the inflamed site stay in the blood vessel! /, Or move to tissues around the vascular force. To do. The scanning microscope uses IV-OB 35F22W20, 1.3 mm diameter lens (OLYMPUS CO., LTD).

[0312] (Olympus IV100 observation)

(Method)

To stain leukocytes and vascular endothelial cells, 150 1 atalidine orange (AO) (0.5%, wZv%) is also administered to the tail vein force of mice just prior to observation. Glycosylated liposomes (K1) or glycosomes without sugar chains are also administered via tail vein force (100 1Z mice). Measurement conditions use the following: Excitation for Cy5.5 and AO is provided by HeNe-R laser (633 nm) and argon laser (488 nm), respectively. Luminescence is captured by Cy5.5 channel (660-730 nm bandpass filter) for Cy5.5, and captured by green fluorescent protein (GFP) channel (505-525 nm bandpass filter) for AO.

[0313] Blood vessels in the inflamed area and surrounding tissues (back of the hind limb) are observed at 10, 30, 6, 24, and 48 hours after injection. 10 and 30 minutes after injection No ribosome adhesion or ribosome aggregation to the blood vessel wall is observed, and only liposome flow in the blood vessel is observed. Partial adhesion to the vessel wall (6 hours after injection) and subsequent migration to the perivascular tissue (24 hours after injection) is observed only on the glycosylated ribosome (K1). At 48 hours after injection, the amount of glycosylated ribosome (K1) accumulated in the tissues surrounding the blood vessels increases compared to 24 hours after injection.

[0314] Ribosomes without sugar chains migrate to the surrounding tissues at 24 and 48 hours after injection, but this accumulation is lower than that of sugar-modified ribosomes (K1). None Liposomal force Indicates passive migration through the vascular endothelium gap caused by inflammation.

[0315] (Example 30. Experiment using tumor-bearing mouse)

(Creation of tumor-bearing mouse model and administration of ribosome)

A mouse having a tumor is produced in the same manner as in Example 23. Glycosylated liposomes (K1) or ribosomes without sugar chains are also administered with tail vein force (200 1 per mouse)

[0316] (Example 31. Imaging with explore Optix)

Using the same method as Example 24, using explore Optix (excitation: 680 nm, emission: 700 nm), the tumor area (back of the hind limb) of the same mouse was examined before administration, 0 hours after administration, and 24 hours. Observe after 48, 72, and 96 hours. Glycomodified ribosomes (K1) accumulate significantly in the tumor area, and this accumulation gradually increases up to 48 hours. On the other hand, the accumulation of ribosomes without sugar chains is constant up to 96 hours, which is lower than the accumulation of sugar chain-modified ribosomes (K1).

[0317] The whole body is scanned 96 hours after injection to examine the distribution of fluorescence in the body. Strong fluorescent signal power Detected in liver, bladder and tumor areas. These signals are analyzed by fluorescence lifetime. Only Cy5.5 bound to HSA is detected in the tumor area. However, it is confirmed in the liver and bladder that both free Cy5.5 and Cy5.5 bound to HSA are present.

[0318] (Example 32. Imaging with a scanning microscope)

(Olympus IV100 observation) (Method)

A method similar to Example 25 is used. Check if the sugar-modified ribosome stays in the blood vessel or if it moves from the blood vessel to the tumor tissue.

[0319] Forty-eight hours after injection, the fluorescent signal also translocates vascular force to the surrounding tissue and is detected in the cells of the tissue. In addition, leukocyte rolling is observed on the vascular endothelium in the tumor area. This indicates that the sugar chain-modified ribosome (K1) was recognized by E-selectin expressed with tumor growth, and as a result, accumulated in the tumor area.

[0320] Ribosomes without sugar chains also accumulate in surrounding tissues of blood vessels, but the amount of this accumulation is less than that of sugar chain-modified ribosomes (K1). The sugar-free ribosome is thought to have passively moved to the vascular endothelium's gap force around the tumor.

[0321] (Summary)

As with fluorescently-encapsulated sugar chain-modified ribosomes, glycosylated ribosomes (K1) modified with SLX also have an affinity for E-selectin and excellent retention in blood vessels. It is thought that it accumulates specifically and efficiently in the inflamed area and tumor tissue. Therefore, the sugar chain-modified ribosome (K1) externally attached to the fluorescent labeling substance is also useful as an in vivo fluorescence imaging reagent in the same manner as the fluorescence-encapsulated sugar chain-modified ribosome.

[0322] (Example 33. In vivo experiment using Cy7-encapsulated sugar chain-modified ribosome)

(Preparation of Cy7-encapsulated sugar chain-modified ribosome)

Cy7-encapsulated sugar chain-modified ribosomes were prepared using the same method as in Examples 1 to 12, except that Cy7 was used instead of Cy5.5 as the fluorescent dye. SLX (K-1) was used as the sugar chain.

[0323] (Production of tumor-bearing mice)

Using the same method as in Example 23, 1 × 10 ⁶ Ehrlich ascite tumor (EAT) cells were transplanted subcutaneously into the right thigh of 6-week-old female BalbZc mice. These tumor-bearing mice were used for experiments after 7-10 days.

[0324] (Evaluation method) Anesthesia was performed by administering 1/10 Nembutal solution into the peritoneal cavity of mice bearing cancer. Next, cy7-encapsulated sugar chain-modified ribosome (K1-3 (50 gZmD SO / zl)) was administered from the tail vein, and image data was collected over time immediately after administration using the fluorescence imaging device eXplore Optix (GE Healthcare). All the image data were taken from the ventral side, and as a control, a tumor-bearing mouse administered with the same amount of ribosome not bound to a sugar chain was used.

[0325] (Result)

Six hours after administration, tumor-bearing mice to which Cy7-encapsulated sugar chain-modified ribosome (K 1) was administered had higher fluorescence intensity at the tumor site than the control. At 24 hours after administration, almost no signal was detected (see Figure 50). O

(Example 34. In vivo experiment using a sugar chain-modified ribosome (Cy3-encapsulated sugar chain-modified ribosome) fluoresced using Cy3)

Cy3-encapsulated sugar chain-modified ribosomes were prepared using the same method as in Examples 1 to 12 except that Cy3 was used instead of Cy5.5 as the fluorescent dye. SLX (K-1) was used as the sugar chain.

[0327] (Production of tumor-bearing mice)

[0328] (Evaluation method)

Anesthesia was performed by administering 1/10 Nembutal solution into the peritoneal cavity of mice bearing cancer. Subsequently, cy3-encapsulated sugar chain-modified ribosome (K-1) (Κ1-3 (50 ^ ₈ / πι L) 100 1) was administered from the tail vein. 48 hours after administration, the tumor site was taken out and fixed with a formalin solution for 12 hours or more, and then a paraffin section was prepared by a conventional method, and image data was obtained with a fluorescence microscope CKX41 (OL YMPUS). As a control, cancer-bearing mice administered with the same amount of liposomes to which no sugar chain was bound were used.

[0329] (Result)

6 hours after administration, the tumor-bearing mice administered Cy3-encapsulated glycosylated ribosome (K 1) It was found that the fluorescence intensity in the tumor tissue was higher than in the control (see Figure 51).

(Example 35. Production of fluorescent dye-containing sugar chain-modified ribosome)

In this example, fluorescent dyes Cy3. Cy5.5 and Cy7 were used as labeling substances, respectively.

3, 3 and 1, dithiobis (sulfosuccinimidyl propionate) (DTSSP; Pierce Co., USA) was dissolved in 500 ml of z / L solution containing carbonate buffer. . 50 L of this solution was added to 1 mL of a solution containing ribosome and stirred at room temperature for 2 hours. Desalting was performed using a centrifugal ultrafiltration filter (NMWL,: 30,000). The solution was concentrated to 100 to 2001, and solution A was added to make 1 ml. This operation was repeated once more.

[0331] (1. Preparation of sugar chain solution to be bound to ribosome)

Sugar chains SLX, N-acetyllactosamine, and α-mannobiose were weighed into glass vials each capable of being sealed and completely dissolved in 500 L of purified water. The final concentration of the sugar chain aqueous solution was adjusted to mM.

[0332] 0.3 g of hydrogen carbonate ammonia was added, and the mixture was stirred at 37 ° C for 3 days, taking care not to dissolve the hydrogen carbonate ammonia. When sodium bicarbonate was completely dissolved, an appropriate amount of ammonium bicarbonate was added. This solution was placed in a refrigerator for 30 minutes and filtered through a 0.45 / z m filter.

[0333] (2. Binding of sugar chain to ribosome and hydrophilization)

A sugar chain solution of 12.5 / zL was added to 1 mL of a solution containing these ribosomes, mixed by vortexing, and reacted at room temperature for 2 hours. Solution B containing Tris buffer was added at 40 / zL, stirred at room temperature for 2 hours, and further stirred at refrigeration (2-10 ° C) for 16-20 hours. Desalination was performed using a centrifugal ultrafiltration filter (NMWL,: 30,000). The desalted solution was concentrated to 100 to 2001, and the solution C containing HEPES buffer was made up to 1 ml. This operation was repeated once more. The solution was filtered through a 0.45 m filter (Myletus—HV (Millipore)) and stored refrigerated under light shielding.

[0334] (3. Measurement of lipid concentration, average particle diameter, Z potential, absorbance, confirmation of stability)

For these ribosomes, protein concentration, lipid concentration, average particle size, zeta potential Was measured. The lipid concentration, average particle diameter, Z potential, absorbance, and stability of the ribosome prepared in this example are the lipid concentration, average particle diameter, Z potential, and absorbance of the sugar chain-modified ribosome prepared in Examples 7-12. , Stability and power. The ribosome prepared by this example was not affected by the sugar chain bound to the surface.

[0335] (3— 1. Protein concentration)

The protein concentration of these sugar chain-modified ribosomes was measured by the same method as in Example 16. As a result, the protein concentration was 0.24 mgZmL or more for Cy3, 0.45 mgZmL or more for Cy5.5, and 0.20 mgZmL or more for Cy7 (see Table 8).

[0336] (3-2. Lipid concentration)

The lipid concentration of these sugar chain-modified ribosomes was measured by the same method as in Example 16. As a result, the lipid concentration was 1.2 mgZmL or higher for Cy3, 1.4 mgZmL or higher for Cy5.5, and 2. lmgZmL or higher for Cy7 (see Table 8).

[0337] (3-3. Average particle size)

The average particle size of these sugar chain-modified ribosomes was measured in the same manner as in Example 16. As a result, the average particle size is 50ηπ when using any fluorescence! ~ 300ηm (see Table 8) o

[0338] (3-4. Zeta potential)

The zeta potential of these sugar chain-modified ribosomes was measured by the same method as in Example 16. As a result, the zeta potential was less than –30 for any fluorescence used (see Table 8).

[0339] (3-5. Stability)

Each manufactured ribosome was refrigerated and stored to confirm the stability. As a result, the particle size distribution is almost the same as that immediately after preparation for at least 1 year for Cy3, for at least 1 year for Cy5.5, and for at least 3 months for Cy7. The characteristics are shown.

[0340] [Table 7] (Table 7. Physical properties of various sugar chain-modified ribosomes)

(Example 36. Improved method for producing sugar chain-modified ribosome (centrifugation))

Using the same method as in Example 1, 10 ml of ribosome (average particle size lOOnm) was prepared.

[0342] (1. Hydrophilization treatment on ribosomal lipid membrane surface)

Ribosome solution prepared in the same way as in Example 1 12.5 ml of Amicon Ultra-4 (fractionated molecular weight: 100,000) (Millipore) 4 aliquots are placed in 4 equal portions, and 2000 X g for 60 minutes The solution was ultrafiltered and concentrated by centrifuging at room temperature. 2 ml of carbonate buffer (CBS buffer: 50 mM NaHCO 157 mM NaCl (pH 8.5)) is added to the ribosome concentrate.

3,

Each was added, and ultrafiltration was performed in a centrifuge under the same conditions. The concentrated ribosome solution was collected and collected, and the same buffer was added to make up the original volume. Next, 12.5 mg of a crosslinking reagent bis (sulfosuccinimidyl) suberate (BS ³ ; Pierce Co., USA) was added, and the mixture was stirred at room temperature for 2 hours. Then, to complete the chemical bonding reaction between the lipid Jiparu Mito I le phosphatidylethanolamine § Min and BS ³ on the ribosome film was stirred overnight further under refrigeration. Liposome solution 12.5 ml Amicon Ultra—4 (molecular weight cut off: 100,000) Add 4 equal aliquots to each of 4 tubes and centrifuge at 2000 X g for 60 minutes at room temperature. The filtrate was concentrated. To this ribosome concentrate, 2 ml each of CBS buffer (pH 8.5) was added, and ultrafiltration was performed in a centrifuge under the same conditions. The concentrated ribosome solution was collected and collected, and the same buffer was diluted to the original volume. Next, 50 mg of tris (hydroxymethyl) aminomethane dissolved in 1.25 ml of CBS buffer (pH 8.5) was added to 12.5 ml of ribosome solution. Next, this solution was stirred at room temperature for 2 hours and then stirred overnight under refrigeration to complete the chemical binding reaction between BS ³ bound to lipids on the ribosome membrane and tris (hydroxymethyl) aminomethane. This ribosome solution is divided into 4 grades for each of 4 Amicon Ultra— 4 (molecular weight cut off: 100,000) Then, it was concentrated by ultrafiltration by centrifuging at 2000 xg for 60 minutes at room temperature. To this ribosome concentrate, 2 ml each of N-tris (hydroxymethyl) -3-amaminopropanesulfonate buffer (PH8.4) was added, and ultrafiltration was performed in a centrifuge under the same conditions. The concentrated ribosome solution was collected and collected, and the buffer was diluted to the original volume.

[0343] (2. Binding of human serum albumin (HSA) to ribosome membrane surface)

Sodium metaperiodate dissolved in 0.3 ml of N-tris (hydroxymethyl) 3-aminopropanesulfonate buffer (pH 8.4) was added to 12.5 ml of the ribosome solution obtained in 1 above. 13. 45 mg was added and stirred with periodate under refrigeration. Amicon Ult ra-4 (fractionated molecular weight: 100,000) Each of the four was divided into 4 equal parts, and concentrated by ultrafiltration by centrifuging at 2000 X g for 60 minutes at room temperature. To this ribosome concentrate, 2 ml of PBS buffer (pH 8.0) was added, and ultrafiltration was performed using a centrifuge under the same conditions. The concentrated ribosome solution was collected and collected, and the volume of the buffer was increased to the original volume.

[0344] To this ribosome solution, 25 mg of human serum albumin (HSA) ZPBS buffer (pH 8.0) was added and allowed to react at room temperature for 2 hours, and further stirred overnight under refrigeration with ganglioside on the ribosome. HSA was bound. Next, each of the four Amicon Ultra-4 (fractionated molecular weight: 100,000) was divided into 4 equal parts, and concentrated by ultrafiltration by centrifuging at 2000 × g for 60 minutes at room temperature. To this ribosome concentrate, 2 ml of CBS buffer (pH 8.5) was added, and ultrafiltration was performed using a centrifuge under the same conditions. The concentrated ribosome solution was collected and collected, and the buffer solution was made up to the original volume to obtain 12.5 ml of HSA-bound ribosome solution.

[0345] Cy7-encapsulated ribosome produced using the same method (conventional method) as in Example 1 except that Cy7 was used instead of Cy5.5 as fluorescence, and the method of this example (centrifugation). The properties of the produced Cy7-encapsulated ribosome were compared. The results are shown in Table 8, Figure 52 and Figure 53 below. According to the method of this example, the yield was improved by about 19% compared to the conventional method, the amount of the buffer used could be saved, and the operation time was shortened. In addition, manufacturing time has been significantly reduced.

[0346] (Table 8. Comparison of lipid concentration, average particle size, Z potential, absorbance) [Table 8]

[0347] As described above, the power that has exemplified the present invention using the preferred embodiment of the present invention. The present invention should not be construed as being limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and literature references cited in this specification should be incorporated by reference as if the contents themselves were specifically described in the present specification. Is understood.

Industrial applicability

[0348] The present invention has utility that a drug can be delivered to a target delivery site by oral administration or the like. Therefore, the present invention provides a drug delivery vehicle for oral administration in which a drug or gene is encapsulated in a sugar chain-modified ribosome and related utility.

Claims

The scope of the claims

[1] A sugar chain-modified ribosome, wherein the sugar chain-modified ribosome is

Ribosome and

Sugar chain and

Linker with protein group

A hydrophilic compound group,

[2] The sugar chain-modified ribosome includes structure I and structure 、,

The structure I is

X-CH NH— R ¹ — NH— C (= 0) — R ² — C (= 0) — NH— R ³

2

R ¹ is the linker protein group,

R ² is a linker-protein cross-linking group,

R ³ is the sugar chain group; and

The structure II is

Y-NH-C (= 0)-R ⁴ -C (= 0)-NH-R ⁵

R ⁴ is the hydrophilic compound crosslinking group,

2. The sugar chain-modified ribosome according to claim 1, wherein R ⁵ is the hydrophilic compound group.

[3] The sugar chain-modified ribosome according to claim 1, wherein the ribosome has fluorescence.

[4] The sugar chain-modified ribosome according to claim 3, wherein the fluorescence is imparted by a fluorescent dye compatible with the ribosome.

[5] The fluorescent dye is

[Chemical 1] Cy5. 5>

[Chemical 2]

<cy3>

cy5, cy7, cy3B, cy3.5, Alexa Fluor 350 ^ Alexa Fluor488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor555, Alexa Fluor568, Alexa Fluor 5 94, Alexa Fluor633, Alexa Fluor647, Alexa Fluor680, Alexa Fluor700, Alexa 5. The sugar chain-modified liposome according to claim 4, which is selected from the group consisting of Fluor 750 and fluorescein-4-isothiocyanate (FITC) and a combination force thereof.

The fluorescent dye is

[Chemical 3] Cy5. 5>

The sugar chain-modified ribosome according to claim 5, wherein

7. The sugar chain-modified liposome according to claim 4, wherein the fluorescent dye is encapsulated in a ribosome.

8. The sugar chain-modified liposome according to claim 2, wherein R ¹ is a mammal-derived protein group.

[9] wherein R ¹ is a human-derived protein groups, glycosylation ribosome of claim 8.

10. The sugar chain-modified ribosome according to claim 9, wherein R ¹ is a human-derived serum protein group.

11. The sugar chain-modified ribosome according to claim 8, wherein R ¹ is a serum albumin group.

[12] The R ² is 3, 3, 1 dithiobis (sulfosuccinimidyl propionate) group, bissulfosuccinimidyl suberate group, disuccinimidyl glutarate group, dithiobis succinate. 3. A group selected from the group consisting of a midylpropionate group, a disuccinimidyl suberate group, an ethylene glycol bissuccinimidyl succinate group and an ethylene glycol bissulfosuccinimidyl succinate group. The sugar chain-modified ribosome described in 1.

[13] The sugar chain-modified ribosome according to claim 12, wherein R ² is a 3, 3, -dithiobis (sulfosuccinimidyl propionate) group.

14. The sugar chain-modified ribosome according to claim 2, wherein R ³ is selected from the group consisting of sialyl Lewis X group, N-acetylyllactosamine group, a 1-6 mannobiose group, and a combination force thereof.

[15] said R ^3, a 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, serial to claim 14 The sugar chain-modified ribosome listed.

[16] The R ³ is an N-acetyllactosamine group, and the N-acetylyllactosamine group is included at a modified bond density in the range of 0.0 OOlmg sugar chain Zmg lipid to 500 mg sugar chain Zmg lipid. The sugar chain-modified ribosome according to claim 14.

[17] R ³ is an α 1-6 mannobiose group, and the α 1-6 mannobiose group is included at a modified bond density in the range of 0.0001 mg sugar chain Zmg lipid to 500 mg sugar chain Zmg lipid. The sugar chain-modified ribosome according to claim 14.

[18] R ⁴ is a bis (sulfosuccinimidyl) suberate group, a disuccinimidyl glutarate group, a dithiobissuccinimidyl propionate group, a disuccinimidyl suberate. Selected from the group 3, 3, 1 dithiobis (sulfosuccinimidyl propionate), ethylene glycol bissuccinimidyl succinate and ethylene glycol bissulfosuccinimidyl succinate Item 3. The sugar chain-modified ribosome according to Item 2.

[19] The R ⁴ is bis (sulfosuccinimidyl I succinimidyl) scan base rate group, glycosylation ribosome of claim 18.

[20] The R ⁵ is a tris (hydroxyalkyl) alkylamino group, a sugar chain modification ribosome of claim 2.

[21] The tris (hydroxyalkyl) alkylamino group is

1 to C hydroxy 6 alkyloxy and alkylamino is C

Claims 20 to 6 which are 1-C alkylamino.

The sugar chain-modified ribosome described. 22. The sugar chain-modified ribosome according to claim 21, which is a non-group.

[23] The sugar chain repair according to claim 2, wherein the functional group a has a —CH 2 C (= 0) —CH— group.

twenty two

Decorated ribosome.

[24] The sugar chain-modified ribosome according to claim 23, wherein X is gandioside.

[25] The sugar chain-modified ribosome according to claim 2, wherein the functional group is an amino group.

26. The sugar chain-modified liposome according to claim 25, wherein Y is phosphatidylethanolamine.

[27] Said ribosomal force dipalmitoylphosphatidylcholine, cholesterol, gangliosi 2. The sugar chain-modified ribosome according to claim 1, wherein the sugar chain-modified ribosome comprises didosyl, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine, and sodium cholate.

[28] The ribosomal force comprising dipalmitoyl phosphatidylcholine, cholesterol, ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine and sodium cholate in a molar ratio of 35: 40: 15: 5: 5: 167, Item 27. The sugar chain-modified ribosome according to Item 27.

[29] R ² is a 3, 3, 1 dithiobis (sulfosuccinimidyl propionate) group,

The sugar chain-modified ribosome according to claim 2, wherein R ³ is selected from the group consisting of a sialyl Lewis X group, an N-acetylyl lactosamine group, an α 1-6 mannobiose group, and a combination force thereof.

[30] The R ³ is selected from the group consisting of sialyl Lewis X group, Ν-acetyl lactosamine group, a 1-6 mannobiose group, and a combination force thereof, and

The sugar chain-modified ribosome according to claim 2, wherein R ⁴ is a bis (sulfosuccinimidyl) suberate group.

[31] The R ² is a 3, 3, 1 dithiobis (sulfosuccinimidyl propionate) group, and the R ³ is a sialyl Lewis X group, an N-acetyllactosamine group, α 1-6 Selected from the group consisting of mannobiose groups and their combination powers, and

[32] including the ribosomal force dipalmitoyl phosphatidylcholine, cholesterol, ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine and sodium cholate,

The sugar chain-modified ribosome according to claim 2, wherein R ⁴ is a bis (sulfosuccinimidyl) suberate group. [33] including the ribosomal force 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, and R ³ is a sialyl Lewis X group, an N-acetyllactosamine group, or a 1-6 mannobiose group. And their combination power

R ⁴ is a bis (sulfosuccinimidyl) suberate group, and

The sugar chain-modified ribosome according to claim 2, wherein R ⁵ is a tris (hydroxymethyl) aminomethane group.

[34] The sugar chain-modified ribosome is

[Chemical 4]

<Cy5. 5>

34. The sugar chain-modified ribosome according to claim 33, which is labeled with.

35. The sugar chain-modified ribosome according to claim 1, wherein in the ribosome, the ratio of protein to lipid is about 0.1 to about 0.5.

[36] The sugar chain-modified ribosome force The maximum size of the particle size distribution of the ribosome is about 80 nm.

2. The sugar chain-modified liposome of claim 1 having a particle size of ˜about 165 nm.

[37] The sugar chain-modified ribosome according to claim 1, wherein the sugar chain-modified ribosome has an average particle size of about 50 nm to about 300 nm.

[38] An imaging agent comprising the sugar chain-modified ribosome according to claim 1. [39] A composition for delivering the substance to a desired site, comprising the sugar chain-modified ribosome according to claim 1 and a substance desired to be delivered.

[40] The composition of claim 39, wherein the desired substance is a diagnostic or research reagent.

41. The composition according to claim 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 force.

[42] The composition of claim 39, for use in molecular or in vivo imaging.

[43] The composition of claim 39, wherein the substance comprises the biological agent.

[44] A pharmaceutical composition further comprising the sugar chain-modified ribosome according to claim 1 and a pharmaceutically active ingredient.

[45] The agent according to claim 44, 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. Pharmaceutical composition.

[46] Use of the sugar chain-modified liposome according to claim 1 for the production of a composition for labeling a desired site.

[47] The use according to claim 46, wherein the desired site is selected from the group consisting of brain, liver, kidney, spleen, lung, spleen, heart, inflammatory site and tumor site force.

[48] A method for labeling a desired site,

Administering a composition for labeling the desired site to the subject, the composition comprising the sugar chain-modified ribosome of claim 1 and a pharmaceutically acceptable carrier, The method wherein the desired site is selected from the group consisting of brain, liver, kidney, spleen, lung, spleen, heart, inflammatory site and tumor site.

[49] A method for producing a sugar chain-modified ribosome, the method comprising:

(b) suspending the lipid membrane in a suspension buffer and sonicating;

(c) providing the fluorescently labeled liposome by mixing the sonicated solution and the fluorescently labeled solution; (d) a step of hydrophilic treatment of the ribosome with tris (hydroxyalkyl) aminoalkane;

[50] The fluorescent labeling solution in the step (c)

[Chemical 5] Ku5. 5>

The method for producing a sugar chain-modified ribosome according to claim 49, comprising:

[51] The production method according to [49], wherein the linker protein strength in the step (e) is human serum albumin.

[52] A method for producing a sugar chain-modified ribosome for delivering a drug to a target delivery site, the production method comprising the following:

(ii) suspending the lipid membrane in a suspension buffer and sonicating;

(iii) a step comprising mixing the sonicated solution and the fluorescent labeling solution (b) determining the density of the sugar chain on the sugar chain-modified ribosome to achieve optimal delivery to the delivery site; and

Manufacturing method.

[53] A method for producing a fluorescent dye-containing sugar chain-modified ribosome,

B) Hydrophilizing the ribosome;

C) binding the ribosome and linker protein; and

D) Step of binding a sugar chain to the ribosome

Manufacturing method.

54. The production method according to claim 53, further comprising a step of performing filter filtration following the step D).

[55] The step A) includes the following:

54. The method for producing a fluorescent dye-containing sugar chain-modified ribosome according to claim 53, comprising:

[56] The fluorescent dye standard in which the fluorescent dye solution in the step (A4) is labeled with a fluorescent dye Is a solution containing a protein, the following:

56. The method of claim 55, which is prepared by a process comprising

[57] The step B) includes the following:

54. The method of claim 53, comprising:

[58] Step B) includes the following:

(B1 ') The solution containing the fluorescent dye or the solution containing the bound ribosome should be separated and dissipated twice under the conditions of a fractional amount of 100,000, 2000 X g, 60 minutes. A step of exchanging the buffer contained in the solution with a carbonate buffer (PH8.5); (Β2 ') Add bis (sulfosuccinimidyl) suberate to the solution in which the buffer solution is converted to the carbonate buffer solution in the step (Bl'), and refrigerate to about The mixture was stirred at 37 ° C and subjected to ultrafiltration by detaching it twice under the conditions of a molecular weight cut off of 100,000, 2000 X g, 60 minutes, and separating, and the free bis (sulfosuccin Imidyl) suberate removal;

54. The method of claim 53, comprising:

Step C) includes the following:

(C4) Further, sodium cyanoboronate ZPBS buffer (pH 8.0) was added to the reaction solution, stirred at refrigeration to room temperature, ultrafiltered with a molecular weight cut off of 300,000, and free Removing sodium boroborate and human serum albumin, and replacing the buffer of the solution with carbonate buffer (PH8.5) 54. The method of claim 53, comprising:

[60] Step C) includes the following:

(C1) Add a solution of sodium metaperoxide ZN tris (hydroxymethyl) -3-aminopropanesulfonate buffer (pH 8.4) to the solution containing the force or bound ribosome containing the fluorescent dye. And acidifying the surface of the ribosome particles by stirring overnight at refrigerated to room temperature;

54. The method of claim 53, comprising:

[61] Step C) includes the following:

(C3 ′) In the step (C2 ′), 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. Melting Producing a liquid;

54. The method of claim 53, comprising:

[62] Step C) includes the following:

(C4 ′) Further, the 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 54. The production method according to claim 53, comprising a step of removing albumin and exchanging the buffer solution of the solution with a carbonate buffer solution (pH 8.5).

[63] Step D) includes the following:

(D2) Add 3, 3 'dithiobis (sulfosuccinimidyl propionate) to the solution containing the force or bound ribosome containing the fluorescent dye, and stir at refrigeration to approximately 37 ° C. And ultrafiltered with a molecular weight cut off of 300,000 to give the free 3,3'-dithiopis (sulfo Removing succinimidyl propionate); and

54. The method of claim 53, comprising:

[64] The production method according to claim 53, further comprising the step of hydrophilicizing the ribosome to which the sugar chain is bound following the step D).

[65] A method for producing a fluorescent dye-containing sugar chain-modified ribosome comprising the following steps:

B) Hydrophilizing the ribosome;

C) binding the ribosome to a linker protein;

D) a step of binding a sugar chain to the ribosome;

E) Hydrophilizing the ribosome to which the sugar chain is bound; and

F) Filtering the solution containing the hydrophilic ribosome

Manufacturing method.

[66] Step A) includes the following:

(A3) A step of stirring the resuspension at 30 to 40 ° C., purging with nitrogen, and sonicating; And

Step B) includes the following:

Step C) includes the following:

(C1) A solution containing sodium phosphoperiodate ZN tris (hydroxymethyl) 3-aminopropanesulfonate buffer solution (PH8.4) is added to a solution containing force or bound ribosome encapsulating the fluorescent dye, Stir overnight under refrigeration to acidify the ribosome particle surface; (C2) In the step (CI), 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 D) includes the following:

(D4) The step (D3) includes a step of exchanging the buffer solution of the solution from which the free sugar chain and the tris (hydroxymethyl) aminomethane are removed with a HEPES buffer solution (pH 7.2). The manufacturing method as described in.

Step A) includes the following:

Step B) includes the following:

(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 (ρΗ8. 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. Removing the carbonate buffer with N-tris (hydroxymethyl) 3-aminopropanesulfonate buffer ( including the step of changing to pH 8.4) to produce a solution containing ribosomes treated with hydrophilicity,

Step C) includes the following:

(C4) Further, the 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) including the step of exchanging,

Step D) includes the following:

(D3) In the step (D2), 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, Sugar chain and Removing the tris (hydroxymethyl) aminomethane; and

Step A) includes the following:

Step B) includes the following:

(B2 ′) Addition of bis (sulfosuccinimidyl) suberate to the solution in which the buffer solution is converted to the carbonate buffer solution in the step (B1 ′) Stir at 37 ° C, ultrafiltration by breaking away twice, separating molecular weight 100,000, 2000 x g for 60 min. And removing the free bis (sulfosuccinimidyl) suberate;

(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. To remove free tris (hydroxymethyl) aminomethane, and replace the carbonate buffer with N-tris (hydroxymethyl) -3-aminopropanesulfonate buffer (pH 8.4). Including a step of producing a solution containing ribosome that has been subjected to hydrophilic treatment, and the step C) includes the following:

(D2) A solution containing ribosome encapsulating or binding the fluorescent dye, Add thiobis (sulfosuccinimidyl propionate), stir at refrigeration to about 37 ° C, ultrafilter with a molecular weight cut off of 300,000, and release the 3, 3, -dithiopis Removing (sulfosuccinimidyl propionate); and

Step A) includes the following:

(Bl ′) The solution containing the fluorescent dye or the solution containing the bound ribosome is limited by separating and separating the solution twice under conditions of a molecular weight cut off of 100,000, 2000 × g for 60 minutes. A step of filtering and replacing the buffer contained in the solution with carbonate buffer (pH 8.5);

(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. To remove free tris (hydroxymethyl) aminomethane, and replace the carbonate buffer with N-tris (hydroxymethyl) -3-aminopropanesulfonate buffer (pH 8.4). Including a step of producing a solution containing ribosomes having been subjected to hydrophilic treatment, wherein the step C) includes the following:

(C4 ′) Further, the reaction solution was stirred at refrigeration to room temperature, and the molecular weight cut off was 100,000, 200 OX g, including ultrafiltration by centrifuging twice for 60 minutes, removing the human serum albumin, and replacing the buffer of the solution with carbonate buffer (pH 8.5) And

Step D) includes the following:

[70] A method for producing a fluorescent dye-containing sugar chain-modified ribosome comprising:

A) forming ribosomes;

B) Hydrophilizing the ribosome;

D) Step of binding a sugar chain to the ribosome

Including the method.

[71] The imaging agent according to claim 38, wherein the sugar chain of the sugar chain-modified ribosome is a sialyl Lewis X group, and the sialyl Lewis X group has a modified bond density of 0.025 mg sugar chain Zmg lipid. . [72] The imaging agent according to claim 71, for imaging an inflamed site or cancer tissue.

73. The imaging agent according to claim 72, wherein the inflammatory site or cancer tissue contains a parenchyma.

74. The composition according to claim 39, wherein the sugar chain of the sugar chain-modified ribosome is a sialyl Lewis X group, and the sialyl Lewis X group has a modified binding density of 0.025 mg sugar chain Z mg lipid.

[75] The composition of claim 74, for delivering the substance to an inflammatory site or cancer tissue

[76] The composition of claim 75, wherein the site of inflammation or cancerous tissue comprises parenchyma.

[77] A carrier for use in molecular imaging or in vivo imaging, the carrier comprising the sugar chain-modified ribosome according to claim 1.

[78] 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 1S 0.025 mg sugar chain Zmg lipid, and the carrier is present in an inflamed site or cancer tissue. 78. A carrier according to claim 77 for delivering a labeling substance.

[79] The carrier of claim 77, wherein the inflammatory site or cancer tissue comprises a parenchyma.

80. The carrier according to claim 79, wherein the labeling substance is fluorescent.

[81] A system for molecular or in vivo imaging of a site of interest comprising:

A) a sugar chain-modified ribosome specific to the target site;

B) sign; and

C) means for examining the presence or absence of the label;

Where, where

system.

82. The system of claim 81, wherein the label is a fluorescent material.

[83] The sugar chain of the sugar chain-modified ribosome is a sialyl Lewis X group, and the sialyl Lewis X group 85. The system of claim 82, comprising 1S 0.025 mg sugar chain Zmg lipid modified binding density.

[84] The system of claim 81, for imaging an inflamed site or cancerous tissue.

[85] The system of claim 84, wherein the site of inflammation or cancerous tissue comprises parenchyma.

[86] The system of claim 81, wherein the means for examining the presence or absence of the label comprises a scanning microscope.

[87] The system of claim 86, wherein the means for checking the presence or absence of a label further comprises a stick objective lens.

[88] The system according to [81], wherein the means for examining the presence or absence of the label is a means for detecting fluorescence.

[89] The method for producing a fluorescent dye-containing sugar chain-modified ribosome according to claim 53, comprising the steps of: a) providing a ribosome encapsulating fluorescence bound by a linker protein; b) making the ribosome hydrophilic The step of chemical conversion treatment;

Including

[90] The method for producing a fluorescent dye-containing sugar chain-modified ribosome according to claim 53, comprising the following steps: a) providing a ribosome encapsulating fluorescence to which a linker protein is bound; b) making the ribosome hydrophilic Process of chemical conversion;

f) Filter the solution containing the hydrophilic dye-containing sugar chain modified ribosome Process

Including

Step c) and step b) are performed in order after step a).

The c) step is as follows:

(cl) adding powder A containing carbonate buffer solution to powder A containing 3, 3, 1 dithiobis (sulfosuccinimidyl propionate) to prepare a mixed solution; and

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) Ammonia bicarbonate (pH 7-14) is added to the aqueous sugar chain solution at a concentration of about 0.2 to 1. Og / mL as necessary, and 3 to 20 to 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;

(d3) The aminated sugar chain solution is added to the solution containing the hydrophilized fluorescently encapsulated ribosome, mixed, and then reacted at room temperature for 2 to 6 hours; a reaction solution step; and ( d4) Solution B containing Tris buffer is added to the reaction solution obtained in step (d3). The mixture is stirred for 2 to 6 hours at room temperature, further stirred for 16 to 48 hours under refrigeration, ultrafiltered with a molecular weight cut off of 30,000, desalted, and modified with the above-mentioned fluorescent dye-containing sugar chain Including the step of producing ribosomes,

Step e)

(el) Concentrate the solution containing the fluorescent dye-containing sugar chain-modified ribosome, and add solution C containing 2- [4- (2 hydroxyxetyl) -1-piperajuryl] ethanesulfonic acid (HEPES) buffer. , Ultrafiltered with a molecular weight cut off of 30,000, concentrated, added the solution C to the concentrated solution containing the fluorescent dye-containing sugar chain-modified ribosome, and the fluorescence-encapsulated type to which the sugar chain was bound Including the step of hydrophilizing the ribosome,

The manufacturing method according to claim 90.

[92] A kit for producing a fluorescent dye-containing sugar chain-modified ribosome,

i) a means for encapsulating or binding the fluorescent dye in the ribosome;

ii) a hydrophilizing agent for the ribosome;

iii) the ribosomal linker protein, and

iv) sugar chains;

V) Means for binding the sugar chain to the ribosome

A kit comprising:

[93] A kit for producing a fluorescent dye-containing sugar chain-modified ribosome comprising the following:

A) a solution containing a ribosome encapsulating fluorescence bound to a linker protein;

B) Powder A containing 3, 3, 1 dithiobis (sulfosuccimidyl propionate) and

C) Solution A containing carbonate buffer and

D) solution B containing Tris buffer and