JP3882034B2 - Intestinal absorption control liposome - Google Patents

Description

【００３８】
【実施例１１】
クロラミンT法による各種糖鎖結合リポソームの¹²⁵I標識
クロラミンT（Wako Pure Chemical Co.,Japan）溶液並びに二亜硫酸ナトリウム溶液をそれぞれ3mg/ml 並びに5mg/ml となるように用事調製して用いた。 （実施例４）から（実施例９）により調製した１３種の糖鎖結合リポソーム並びにtris(hydroxymethyl)aminomethane結合リポソームとを50μl ずつ別々にエッペンチューブに入れ、続いて¹²⁵I-NaI（NEN Life Science Product,Inc.USA）を15μl、クロラミンT溶液を10μl 加え反応させた。5 分ごとにクロラミンT溶液10μl を加え、この操作を2 回繰り返した後15 分後に還元剤として二亜硫酸ナトリウム100μl 加え、反応を停止させた。次に、Sephadex G-50（Phramacia Biotech.Sweden）カラムクロマト上に乗せ、PBS で溶出、標識体を精製した。最後に、非標識-リポソーム複合体を添加して比活性（4 x 10⁶ Bq/mg protein）を調整して１３種類の¹²⁵I標識リポソーム液を得た。
【００３９】
【実施例１２】
各種の糖鎖結合リポソーム複合体のマウスでの腸管から血中への移行量の測定
一昼夜水分以外絶食した雄性ddYマウス（7週齢）に、（実施例１１）により¹²⁵I標識された１３種の糖鎖結合並びにtris(hydroxymethyl)aminomethane結合リポソーム複合体0.2mlを蛋白質量として3μg/一匹の割合になるように、マウス用経口ゾンデで腸管内に強制投与した後、10分後にネンブタール麻酔下で下大動脈より血液1mlを採血した。そして、血中の¹²⁵I放射能をガンマーカウンター(Alola ARC300)で測定した。さらに、各種のリポソーム複合体の生体内安定性を調べる目的で、各血液の血清をSephadex G-50で再クロマトしたが、いずれも大半の放射能が高分子量のボイドフラクションにみられ、各種のリポソーム複合体は生体内においても安定性を有していた。なお、腸管から血中への放射能移行量は、投与全放射能に対する血液1ml当たりの放射能の割合（％投与量/ml血液）で表示した。この結果を図６から図９に示す。
【００４０】
【発明の効果】
本発明の上記糖鎖修飾リポソームは腸管吸収性に優れ、腸管を経由するという、従来のリポソーム使用製剤にはみられない新たな投与形態で投与可能なものであるという点で画期的なものである。さらに、腸管吸収性は、リポソームに対する糖鎖結合量の設定と、糖鎖の選択によって制御することができ、これにより、効率的、かつ副作用がなく安全に、薬剤あるいは遺伝子等を腸管経由で生体組織に移行することが可能となり、医学、薬学分野において特に有用なものである。
【図面の簡単な説明】
【図１】は、ラクトース二糖鎖により修飾されたリポソームの構造例を示す模式図である。
【図２】は、2’-フコシルラクトース三糖鎖により修飾されたリポソームの構造例を示す模式図である。
【図３】は、ジフコシルラクトース四糖鎖により修飾されたリポソームの構造例を示す模式図である。
【図４】は、3-フコシルラクトース三糖鎖により修飾されたリポソームの構造例を示す模式図である。
【図５】は、比較試料としてtris(hydroxymethyl)aminomethaneを結合したリポソームの模式図である。
【図６】は、ラクトース二糖鎖の結合量を変えた３種のリポソーム複合体の腸管内投与１０分後の血中への移行量をコントロールとともに示す図である。
【図７】は、2’-フコシルラクトース三糖鎖の結合量を変えた３種のリポソーム複合体の腸管内投与１０分後の血中への移行量をコントロールとともに示す図である。
【図８】は、ジフコシルラクトース四糖鎖の結合量を変えた３種のリポソーム複合体の腸管内投与１０分後の血中への移行量をコントロールとともに示す図である。
【図９】は、3-フコシルラクトース三糖鎖の結合量を変えた３種のリポソーム複合体の腸管内投与１０分後の血中への移行量をコントロールとともに示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention can be applied in the medical and pharmaceutical fields including pharmaceuticals and cosmetics, recognizes a target cell / tissue such as cancer, and locally delivers a drug or gene to an affected area. The present invention relates to a sugar chain-modified liposome that can be used as a cell / tissue sensing probe, and particularly excellent in intestinal absorption, and a liposome preparation in which a drug or gene is encapsulated.
[0002]
[Prior art]
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) that targets cancer cells and target tissues” was listed. In the Nanotechnology / Materials Promotion Strategy of the Japan Science and Technology Council, there is “Nanobiology that uses and controls the medical microsystems / materials / biological mechanisms” as one of the priority areas. One example is “Establishing basic seeds for biofunctional materials and pinpoint treatment for extending healthy life expectancy”. On the other hand, with the aging of society, the incidence and death rates of cancer are increasing year by year, and the development of target-oriented DDS, which is a novel therapeutic material, is awaited. The importance of target-oriented DDS nanomaterials with no side effects in other diseases is drawing attention, and the market size is predicted 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 manifested by the drug reaching a specific target site and acting there. On the other hand, a side effect caused by a drug is that a drug acts on an unnecessary site. Therefore, development of a drug delivery system is also required in order to use the drug effectively and safely. Among them, in particular, targeting DDS is a concept of delivering a drug “to a necessary site in the body”, “a necessary amount”, and “only for a necessary time”. Liposomes, which are fine particle carriers, are attracting attention as a representative material for that purpose. Passive targeting methods such as changing the lipid type, composition ratio, particle size, and surface charge of liposomes have been attempted in order to give these particles a target-directing function. There is a need for improvements.
[0004]
On the other hand, active targeting methods have also been attempted to enable highly functional targeting. This is an ideal targeting method, also called "missile drug", but it has not been completed in Japan and overseas, and future development is highly expected. In this method, a ligand is bound on the surface of the liposome membrane, and the receptor present on the cell membrane surface of the target tissue is specifically recognized, thereby enabling targeting actively. As a ligand for 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 and glycoprotein sugar chains are information molecules in various cell-to-cell communications such as generation and morphogenesis of biological tissues, cell proliferation and differentiation, biological defense and fertilization mechanisms, canceration and metastasis mechanisms. It is becoming clear that it plays an important role.
[0005]
In addition, research on various lectins (glycan recognition proteins) such as selectin, siglec, and galectin as receptors existing on the cell membrane surface of each target tissue has advanced, and thus has various molecular structures. Sugar chains have attracted attention as new DDS ligands (1) Yamazaki, N., Kojima, S., Bovin, NV, Andre, S., Gabius, S. and Gabius, H.-J. (2000) Adv. Drug Delivery Rev.43, 225-244. (2) Yamazaki, N., Jigami, Y., Gabius, H.-J., Kojima, S (2001) Trends in Glycoscience and Glycotechnology13, 319-329.http://www.gak.co.jp/TIGG/71PDF/yamazaki.pdf).
[0006]
With regard to liposomes having a ligand bound to the outer membrane surface, many studies have been made as DDS materials for selectively delivering drugs or genes to target sites such as cancer. However, most of them bind to target cells in vitro but are not targeted to target cells and tissues expected in vivo ((1) Forssen, E. and Willis, M. (1998) Adv Drug Delivery Rev.29249-271. (2) Toshio Takahashi and Mitsuru Hashida (1999), Today's DDS / Drug Delivery System, pages 159-167, Pharmaceutical Journal, Osaka). In research and development of DDS materials that utilize the molecular recognition function of sugar chains, some studies have been made on liposomes into which glycolipids having sugar chains have been introduced. Their function evaluation is performed in vitro. However, research on liposomes into which glycoproteins having sugar chains have been introduced has hardly progressed ((1) DeFrees, SA, Phillips, L., Guo, L. and Zalipsky, S. (1996) J. Am Chem. Soc.118(2) Spevak, W., Foxall, C., Charych, D.H., Dasqupta, F. and Nagy, J.O. (1996) J. Med. Chem.39, 1018-1020. (3) Stahn, R., Schafer, H., Kernchen, F. and Schreiber, J. (1998) Glycobiology8, 311-319. (4) Yamazaki, N., Jigami, Y., Gabius, H.-J., Kojima, S (2001) Trends in Glycoscience and Glycotechnology13, 319-329.http://www.gak.co.jp/TIGG/71PDF/yamazaki.pdf). Therefore, systematic research including preparation methods and in vivo kinetics (in vivo) analysis of liposomes bound to a wide variety of glycolipids of glycolipids and glycoproteins has not been developed so far, and future progress is expected. This is an important issue.
Furthermore, as a new type of DDS material research, the development of DDS materials that can be used by oral administration, which can be administered most simply and inexpensively, is also an important issue. For example, peptide drugs and the like are generally water-soluble and have a high molecular weight, and the gastrointestinal tract has a low permeability to the small intestinal mucosa, so that they are hardly absorbed in the intestinal tract even if administered orally due to enzymatic degradation. Therefore, research on ligand-bound liposomes has been attracting attention as a DDS material for delivering these high molecular weight drugs and genes from the intestinal tract into the blood (Lehr, C.-M. (2000) J. Controlled Release).65, 19-29). However, studies on intestinal absorption-controllable liposomes using sugar chains as these ligands have not yet been reported.
[0007]
[Problems to be solved by the invention]
Accordingly, an object of the present invention is to provide a sugar chain-modified liposome complex that has specific binding activity to various lectins (sugar chain recognition proteins) present on the cell surface of various tissues and that is particularly excellent in intestinal absorbability. It is to provide a liposome preparation in which a drug and a gene are encapsulated in the body and the liposome.
[0008]
[Means for Solving the Problems]
  In order to solve the above problems, as a result of intensive studies, the present inventors have found that liposomes whose surface is modified with a specific sugar chain are extremely excellent in intestinal absorbability, and can be applied to various tissues of drugs and genes in vivo. It has been found that the preparation is useful in transportation, and the present invention has been completed.
  That is, the present invention relates to the following (1) to (7).
(1) Lactose disaccharide chain, 2'-fucosyl lactose trisaccharide chain, difucosyl lactoseTetrasaccharide chainAnd liposomes modified with sugar chains selected from 3-fucosyl lactose trisaccharide chainsA liposome characterized in that the sugar chain is bound to a liposome membrane via a linker protein.
(2) The liposome according to (1) above, wherein the liposome surface and / or linker protein is made hydrophilic.
(3)The above (the liposome surface and / or the linker protein is made hydrophilic by tris (hydroxymethyl) aminomethane (2)Liposomes described
(4)Linker protein is human serum albuminOr any one of said (1)-(3) which is bovine serum albuminThe liposome according to 1.
(5)The above-mentioned liposome having intestinal absorption function (1) to any one of (4)The liposome described.
(6)The above (adjusted amount of sugar chain on the liposome membrane (1) to (5)The liposome according to any one of the above.
(7) Above (A liposome preparation in which a drug or gene is encapsulated in the liposome according to any one of 1) to (6).
[0009]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in more detail.
Liposomes usually mean closed vesicles composed of a lipid layer assembled in a membrane and an aqueous layer inside. The liposome in the present invention comprises lactose disaccharide chain (Gal.beta.1-4Glc), 2′-fucosyl lactose trisaccharide chain (Fuc.alpha.1-2Gal.beta.1-4Glc), difucosyl lactose tetrasaccharide chain (Fuc.alpha.1-2Gal.beta.1-4 (Fuc.alpha.1-3) Glc) and 3-fucosyl lactose trisaccharide (Gal.beta.1-4 (Fuc.alpha.1-3) Glc) is modified with a sugar chain selected from. These sugar chains are commercially available.
In the present invention, these sugar chains are preferably bound to the liposome surface via a linker protein. The structure of the liposome in this case is shown in FIGS. 1 to 4 together with the chemical structure of these sugar chains. These figures are schematic diagrams, and many sugar chain-linker proteins are actually bound to the liposome surface.
Examples of the linker protein include animal serum albumin such as human serum albumin (HSA) and bovine serum albumin (BSA). In particular, when human serum albumin is used, it is often the case that mouse tissue has a large uptake into each tissue. It is confirmed by the experiment about.
[0010]
Examples of the lipid constituting the liposome of the present invention include phosphatidylcholines, phosphatidylethanolamines, phosphatidic acids, gangliosides or glycolipids or phosphatidylglycerols, cholesterol, and the like. As phosphatidylcholines, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, etc., and as phosphatidylethanolamines, dimyristoyl phosphatidylethanolamine, Dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, etc. include phosphatidic acids such as dimyristoyl phosphatidic acid, dipalmitoyl phosphine. Thydic acid, distearoyl phosphatidic acid, dicetyl phosphoric acid, etc. are gangliosides, ganglioside GM1, ganglioside GD1a, ganglioside GT1b, etc., and glycolipids are galactosylceramide, glucosylceramide, lactosylceramide, sulfatide, globoside As the phosphatidylglycerols, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol and the like are preferable.
[0011]
The liposome used in the present invention may be a normal one, but it is desirable that its surface be made hydrophilic.
[0012]
The liposome itself can be produced according to a well-known method, and examples thereof include a thin film method, a reverse layer evaporation method, an ethanol injection method, and a dehydration-rehydration method.
[0013]
Moreover, it is also possible to adjust the particle diameter of the liposome using an ultrasonic irradiation method, an extrusion 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, gangliosides or glycolipids, or phosphatidylglycerol. A mixed micelle of a lipid and a surfactant sodium cholate is prepared.
[0014]
In particular, the formulation of phosphatidylethanolamines is essential as a hydrophilization reaction site, and the formulation of gangliosides or glycolipids or phosphatidylglycerols is essential as a linker protein binding site. And a liposome is produced by performing ultrafiltration of the mixed micelle obtained by this. Subsequently, the liposome surface is hydrophilized using a divalent reagent for crosslinking and tris (hydroxymethyl) aminomethane on the lipid phosphatidylethanolamine of the liposome membrane.
[0015]
Hydrophilization of liposomes is achieved by a conventionally known method, for example, a method of preparing liposomes using a phospholipid obtained by covalently bonding polyethylene glycol, polyvinyl alcohol, maleic anhydride copolymer or the like (Japanese Patent Laid-Open No. 2010-302686). In the present invention, it is particularly preferable to make the liposome surface hydrophilic using tris (hydroxymethyl) aminomethane.
[0016]
The method using tris (hydroxymethyl) aminomethane of the present invention is preferable in several respects as compared with the conventional hydrophilization method using polyethylene glycol or the like. For example, in the case where a sugar chain is bound on a liposome as in the present invention and its molecular recognition function is utilized for target orientation, 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 particularly preferable because it is less likely to cause steric hindrance to the sugar chain and does not hinder the progress of the sugar chain molecule recognition reaction by the lectin (sugar chain recognition protein) on the target cell membrane surface.
[0017]
In addition, the liposome 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. Is preferable.
[0018]
In order to make the liposome surface hydrophilic using tris (hydroxymethyl) aminomethane, for example, lipids such as dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, and distearoyl phosphatidylethanolamine are used. Bissulfosuccinimidyl suberate, disuccinimidyl glutarate, dithiobissuccinimidyl propionate, disuccinimidyl suberate, 3,3'-dithiobissulfate Dipalmitoyl phosphate on the liposome membrane by reacting with a divalent reagent such as fosuccinimidyl propionate, ethylene glycol bissuccinimidyl succinate, ethylene glycol bissulfosuccinimidyl succinate, etc. Zirethanol A divalent reagent is bound to a lipid such as min, and then tris (hydroxymethyl) aminomethane is reacted with one of the bonds of the divalent reagent to bind tris (hydroxymethyl) aminomethane to the liposome surface. .
[0019]
Specifically, the means for modifying liposomes with sugar chains in the present invention will be described. For example, sugar chains are bound to liposomes via linker proteins. As means for binding linker proteins to liposomes, first, liposomes are treated with NaIO.₄, Pb (O₂CCH₃)₄, NaBiO₃To oxidize gangliosides present on the liposome membrane surface, and then NaBH₃CN, NaBH₄The linker protein and the ganglioside on the liposome membrane surface are bound by a reductive amination reaction using a reagent such as
[0020]
This linker protein is also preferably made hydrophilic, in which a compound having a hydroxy group is bound to the linker protein. For example, bissulfosuccinimidyl suberate, disuccinimidyl glutarate, dithiobissk Cynimidyl propionate, disuccinimidyl suberate, 3,3'-dithiobissulfosuccinimidyl propionate, ethylene glycol bissuccinimidyl succinate, ethylene glycol bissulfosuccinimidyls What is necessary is just to couple | bond tris (hydroxymethyl) aminomethane with the linker protein on a liposome using bivalent reagents, such as a succinate. Specifically, first, one end of a divalent reagent for crosslinking is bound to all amino groups of the linker protein. A sugar chain glycosylamine compound obtained by subjecting the reducing end of each sugar chain to a glycosyl amination reaction is prepared, and the amino group of this sugar chain and another part of the cross-linked divalent reagent bonded above on the liposome are prepared. Join the unreacted end. Next, hydrophilic treatment is carried out using the unreacted end of most of the divalent reagent remaining unreacted with no sugar chain attached to the surface of the protein on the sugar chain-bound liposome membrane thus obtained. Do. In other words, the liposome of the present invention can be obtained by performing a binding reaction between the unreacted end of the divalent reagent bound to the protein on the liposome and tris (hydroxymethyl) aminomethane to make the liposome surface hydrophilic. it can.
[0021]
Hydrophilization of the liposome surface and the linker protein improves the migration to various tissues and the retention in various tissues. This is because when the liposome surface and the linker protein surface are rendered hydrophilic, the portions other than sugar chains appear to be water in the body in each tissue, etc. This is probably due to the fact that only the sugar chain is recognized by the lectin (sugar chain recognition protein) of the target tissue.
[0022]
Next, the sugar chain is bound to the linker protein on the liposome. For example, first, the reducing end of the saccharide constituting the sugar chain is NH₄HCO₃, NH₂COONH₄Glycosylamination using ammonium salts such as bissulfosuccinimidyl suberate, disuccinimidyl glutarate, dithiobis succinimidyl propionate, disuccinimidyl suberate, 3,3 ′ -Dithiobissulfosuccinimidyl propionate, ethylene glycol bissuccinimidyl succinate, ethylene glycol bissulfosuccinimidyl succinate and other divalent reagents were used to bind on the liposome membrane surface The linker protein and the glycosylated amino sugar are bound to obtain a liposome as shown in FIGS.
[0023]
Moreover, although the liposome of the present invention generally has extremely high intestinal absorbability, it further controls the intestinal absorbability by adjusting the density of sugar chains on the liposome, and more efficiently delivers the drug to the target site. Can be transferred, and side effects can be reduced. For example, FIGS. 6 to 9 in the examples show the transferability from the intestinal tract to the blood (that is, intestinal absorbability) when the sugar chain binding amount in the four types of sugar chain-modified liposomes is changed to three stages. .
[0024]
The amount of sugar chain binding was changed by binding sugar chains to linker protein-bound liposomes at three levels (1) 50 μg, (2) 200 μg, (3) 1 mg). It is. According to this, in the case of lactose disaccharide chain, the intestinal absorbability decreases gradually as the sugar chain density increases, but in the case of 2'-fucosyl lactose trisaccharide chain and difucosyl lactose tetrasaccharide chain, the intestinal absorbability Conversely rises.
[0025]
In addition, the 3-fucosyl lactose trisaccharide chain once decreases and increases. These facts indicate that the intestinal absorbability varies with the type of sugar chain depending on the amount of sugar chains bound on the liposome. Therefore, intestinal absorbability can be controlled by appropriately setting the amount of sugar chain bound on the liposome for each type of sugar chain.
[0026]
In order to encapsulate a drug such as an anticancer drug or a gene for gene therapy in the sugar chain-modified liposome of the present invention, a well-known method may be used. For example, a solution containing a drug or the like, phosphatidylcholine, phosphatidylcholine, etc. By forming liposomes using lipids of fatidylethanolamines, drugs and the like are encapsulated in the liposomes.
[0027]
Hereinafter, the feasibility of the present invention will be specifically described with reference to examples. However, the present invention is not limited to these examples.
[0028]
[Example 1]
Preparation of liposomes
Liposomes have been reported in a previously reported method (Yamazaki, N., Kodama, M. and Gabius, H.-J. (1994) Methods Enzymol.242, 56-65), using a modified cholate dialysis method. That is, dipalmitoyl phosphatidylcholine, cholesterol, dicetyl phosphate, ganglioside and dipalmitoyl phosphatidylethanolamine in a molar ratio of 35: 40: 5: 15: 5, respectively, to a total lipid content of 45.6 mg and cholic acid 46.9 mg of sodium was added and dissolved in 3 ml of chloroform / methanol solution. The solution was evaporated and the precipitate was dried in vacuo to obtain a lipid membrane. The obtained lipid membrane was suspended in 3 ml of TAPS buffer (pH 8.4) and sonicated to obtain a transparent micelle suspension. Furthermore, the micelle suspension was subjected to ultrafiltration using a PM10 membrane (Amicon Co., USA) and PBS buffer (pH 7.2) to prepare 10 ml of uniform liposomes (average particle size 100 nm).
[0029]
[Example 2]
Hydrophilization treatment on the liposomal lipid membrane surface
The liposome solution 10 ml prepared in Example 1 was subjected to ultrafiltration using an XM300 membrane (Amicon Co., USA) and a CBS buffer (pH 8.5) to adjust the pH of the solution to 8.5. Next, the crosslinking reagent bis (sulfosuccinimidyl) suberate (BS^ThreePierce Co., USA) 10 ml was added and stirred at 25 ° C. for 2 hours. Then, further stirring overnight at 7 ° C, lipid dipalmitoylphosphatidylethanolamine and BS on the liposome membrane^ThreeThe chemical bonding reaction with was completed. This liposome solution was subjected to ultrafiltration with an XM300 membrane and a CBS buffer (pH 8.5). Next, add 40 mg of tris (hydroxymethyl) aminomethane dissolved in 1 ml of CBS buffer (pH 8.5) to 10 ml of liposome solution, stir at 25 ° C. for 2 hours, and then at 7 ° C. overnight to form lipids on the liposome membrane. Combined BS^ThreeCompleted the chemical bonding reaction between tris (hydroxymethyl) aminomethane and tris (hydroxymethyl) aminomethane. As a result, the hydroxyl group of tris (hydroxymethyl) aminomethane was coordinated on the lipid dipalmitoylphosphatidylethanolamine of the liposome membrane to make it hydrated and hydrophilic.
[0030]
[Example 3]
Binding of human serum albumin (HSA) to the liposome membrane surface
Previously reported methods (Yamazaki, N., Kodama, M. and Gabius, H.-J. (1994) Methods Enzymol.24256-65) using a coupling reaction method. That is, this reaction is a two-step chemical reaction. First, sodium metaperiodate in which ganglioside present on the surface of 10 ml of liposome membrane obtained in Example 2 was dissolved in 1 ml of TAPS buffer (pH 8.4). After adding 43 mg and stirring at room temperature for 2 hours to oxidize periodate, 10 ml of oxidized liposomes were obtained by ultrafiltration with XM300 membrane and PBS buffer (pH 8.0). To this liposome solution, 20 mg of human serum albumin (HSA) was added and stirred at 25 ° C. for 2 hours, then 2M NaBH in PBS (pH 8.0)_Three100 μl of CN was added and stirred overnight at 10 ° C., and HSA was bound by a coupling reaction between ganglioside on liposome and HSA. After ultrafiltration with XM300 membrane and CBS buffer (pH 8.5), 10 ml of HSA-bound liposome solution was obtained.
[0031]
[Example 4]
Binding of lactose disaccharide chain onto liposome membrane surface bound human serum albumin (HSA) (Three types with different sugar chain binding amount)
Lactose disaccharide chain (Wako Pure Chemical Co., Japan) (1) 50μg or 2) 200μg or 3) 1mg 0.25g NH_FourHCO_ThreeAfter stirring at 37 ° C for 3 days, filtration through a 0.45 μm filter completes the amination reaction of the reducing end of the sugar chain to obtain 50 μg of a glycosylamine compound of lactose disaccharide chain. It was. Next, 1 mg of the crosslinking reagent 3,3′-dithiobis (sulfosuccinimidyl propionate (DTSSP; Pierce Co., USA)) was added to 1 ml of a part of the liposome solution obtained in (Example 3), followed by 2 hours at 25 ° C. The mixture was stirred overnight at 0 ° C. and ultrafiltered with XM300 membrane and CBS buffer (pH 8.5) to obtain 1 ml of liposomes in which DTSSP was bound to HSA on the liposomes. Add 50 μg of the chain glycosylamine compound, stir at 25 ° C. for 2 hours, then stir at 7 ° C. overnight, ultrafilter with XM300 membrane and PBS buffer (pH 7.2), and bind to liposome membrane surface bound human serum albumin Lactose disaccharide chains were linked to the above DTSSP, and as a result, three types of lactose disaccharide chains, human serum albumin, and liposomes shown in Fig. 1 were combined (abbreviation: abbreviation: (1) LAC-1, (2) LAC-2, (3) LAC-3) 2ml each (total lipid Amount 2 mg, total protein amount 200 μg, average particle size 100 nm).
[0032]
[Example 5]
Binding of 2'-fucosyl lactose trisaccharide to liposome membrane surface bound human serum albumin (HSA) (Three types with different amounts of sugar chain binding)
2'-fucosyl lactose trisaccharide chain (Wako Pure Chemical Co., Japan) (1) 50μg or 2) 200μg or 3) 1mg is added to 0.25g NH_FourHCO_ThreeAfter stirring at 37 ° C for 3 days, the mixture is filtered through a 0.45 µm filter to complete the amination reaction of the reducing end of the sugar chain, and the glycosylamine of the 2'-fucosyl lactose trisaccharide chain 50 μg of compound was obtained. Next, 1 mg of the crosslinking reagent 3,3′-dithiobis (sulfosuccinimidyl propionate (DTSSP; Pierce Co., USA)) was added to 1 ml of a part of the liposome solution obtained in (Example 3), followed by 2 hours at 25 ° C. The mixture was stirred overnight at 0 ° C., and ultrafiltered with XM300 membrane and CBS buffer (pH 8.5) to obtain 1 ml of liposome in which DTSSP was bound to HSA on the liposome. Add 50 μg of fucosyllactose trisaccharide glycosylamine compound, stir at 25 ° C. for 2 hours, then stir at 7 ° C. overnight, ultrafilter with XM300 membrane and PBS buffer (pH 7.2), and liposome membrane surface 2'-fucosyl lactose trisaccharide chain was conjugated to DTSSP on conjugated human serum albumin, and as a result, 3 kinds of 2'-fucosyl lactose trisaccharide chain shown in Fig. 2 and humans with different sugar chain binding amount Liposomes in which serum albumin and liposomes are bound (abbreviations: (1) 2FL-1, (2) 2FL- 2, 3) 2FL-3) 2 ml each (total lipid amount 2 mg, total protein amount 200 μg, average particle size 100 nm) was obtained.
[0033]
[Example 6]
Binding of difucosyl lactose tetrasaccharide chain to liposome membrane surface bound human serum albumin (HSA) (Three types with different sugar chain binding amount)
Difucosyl lactose tetrasaccharide chain (Wako Pure Chemical Co., Japan) (1) 50μg or (2) 200μg or (3) 1mg) 0.25g NH_FourHCO_ThreeAfter stirring at 37 ° C for 3 days, filtration through a 0.45 μm filter completes the amination reaction of the reducing end of the sugar chain, and 50 μg of the glycosylamine compound of the difucosyl lactose tetrasaccharide chain Got. Next, 1 mg of the crosslinking reagent 3,3′-dithiobis (sulfosuccinimidyl propionate (DTSSP; Pierce Co., USA)) was added to 1 ml of a part of the liposome solution obtained in (Example 3), followed by 2 hours at 25 ° C. The mixture was stirred overnight at 0 ° C., and ultrafiltered with an XM300 membrane and CBS buffer (pH 8.5) to obtain 1 ml of liposomes in which DTSSP was bound to HSA on the liposomes. Add 50μg of tetrasaccharide glycosylamine compound, stir at 25 ° C for 2 hours, then stir overnight at 7 ° C, ultrafilter with XM300 membrane and PBS buffer (pH 7.2) and bind to liposome membrane surface bound human Difucosyl lactose tetrasaccharide chains were bound to DTSSP on serum albumin, and as a result, three types of difucosyl lactose tetrasaccharide chains, human serum albumin and liposomes shown in Fig. 3 were produced. Bound liposome (abbreviation: (1) DFL-1, (2) DFL-2, 3 ▼ DFL-3) each 2 ml (total lipid amount 2 mg, total protein mass 200 [mu] g, average particle size 100 nm) was obtained.
[0034]
[Example 7]
Binding of 3-fucosyl lactose trisaccharide to liposome membrane-bound human serum albumin (HSA) (Three types with different amounts of sugar chain binding)
3-fucosyl lactose trisaccharide (Wako Pure Chemical Co., Japan) (1) 50 μg, or (2) 200 μg, or (3) 1 mg) to 0.25 g NH_FourHCO_ThreeThe solution is added to a 0.5 ml aqueous solution in which the solution is dissolved and stirred at 37 ° C. for 3 days, and then filtered through a 0.45 μm filter to complete the amination reaction of the reducing end of the sugar chain, thereby completing the glycosylamine compound of 3-fucosyl lactose trisaccharide 50 μg was obtained. Next, 1 mg of the crosslinking reagent 3,3′-dithiobis (sulfosuccinimidyl propionate (DTSSP; Pierce Co., USA)) was added to 1 ml of a part of the liposome solution obtained in (Example 3), followed by 2 hours at 25 ° C. The mixture was stirred overnight at 0 ° C. and ultrafiltered with XM300 membrane and CBS buffer (pH 8.5) to obtain 1 ml of liposomes in which DTSSP was bound to HSA on the liposomes. Add 50μg of glycosylamine compound of lactose trisaccharide chain, stir at 25 ° C for 2 hours, then stir at 7 ° C overnight, ultrafilter with XM300 membrane and PBS buffer (pH 7.2) and bind to liposome membrane surface 3-fucosyl lactose trisaccharide chain was bound to DTSSP on human serum albumin, and as a result, three types of 3-fucosyl lactose trisaccharide chain shown in Fig. 4 and human serum albumin Liposomes bound with liposomes (abbreviations: (1) 3FL-1, (2) 3FL-2, ▼ 3FL-3) each 2 ml (total lipid amount 2 mg, total protein mass 200 [mu] g, average particle size 100 nm) was obtained.
[0035]
[Example 8]
Binding of tris (hydroxymethyl) aminomethane onto liposomal membrane-bound human serum albumin (HSA)
In order to prepare liposomes as comparative samples, 1 mg of the crosslinking reagent 3,3′-dithiobis (sulfosuccinimidyl propionate (DTSSP; Pierce Co., USA)) was added to 1 ml of a part of the liposome solution obtained in Example 3 at 25 ° C. The mixture was stirred for 2 hours and then overnight at 7 ° C., and ultrafiltered with an XM300 membrane and CBS buffer (pH 8.5) to obtain 1 ml of liposomes in which DTSSP was bound to HSA on the liposomes. Add 13 mg of tris (hydroxymethyl) aminomethane (Wako Co., Japan), stir at 25 ° C for 2 hours, then stir overnight at 7 ° C, and ultrafilter with XM300 membrane and PBS buffer (pH 7.2). Tris (hydroxymethyl) aminomethane was bound to DTSSP on liposomal membrane-bound human serum albumin, and 13 mg of tris (hydroxymethyl) aminomethane, which is already in large excess, was present in this process. The hydrophilization treatment on HSA) was completed at the same time, and as a result, the final product was made hydrophilic. 2 ml of liposome (abbreviation: TRIS) as a comparative sample in which tris (hydroxymethyl) aminomethane, human serum albumin, and liposome shown in FIG. 5 are bound (total lipid amount 2 mg, total protein amount 200 μg, average particle size 100 nm) was gotten.
[0036]
[Example 9]
Hydrophilization treatment on liposome membrane surface-bound human serum albumin (HSA) For liposomes to which 12 kinds of sugar chains prepared by the means of Examples 4 to 7 were bound, HSA on the liposome was separately treated by the following procedure. The protein surface was hydrophilized. Separately add 13 mg of tris (hydroxymethyl) aminomethane to 2 ml of 12 kinds of sugar chain-bound liposomes, stir at 25 ° C for 2 hours, and then at 7 ° C overnight, then with XM300 membrane and PBS buffer (pH 7.2) The unreacted product is removed by ultrafiltration, and 12 kinds of sugar chain-bound liposome complexes (abbreviations: LAC-1, LAC-2, LAC-3, 2FL-1, 2FL-2, 2FL-3, DFL-1, DFL-2, DFL-3, 3FL-1, 3FL-2, 3FL-3) 2 ml each (total lipid amount 2 mg, total protein amount 200 μg, average particle size 100 nm) Got.
[0037]
[Example 10]
Measurement of lectin binding activity inhibitory effect by various sugar chain-bound liposome complexes In vitro lectin binding activity of each of the 12 sugar chain-bound liposome complexes prepared by the means of Examples 4 to 7 and Example 9, Conventional method (Yamazaki, N. (1999) Drug Delivery System,14, 498-505) in an inhibition experiment using a lectin-immobilized microplate. That is, lectin (E-selectin; R & D Systems Co., USA) was solidified on a 96-well microplate. On this lectin-immobilized plate, 0.1 μg of biotinylated fucosylated fetuin, a comparative ligand, and various sugar chain-bound liposome complexes with different concentrations (0.01 μg, 0.04 μg, 0.11 μg, 0.33 μg, 1 μg as protein mass) ) And incubated at 4 ° C. for 2 hours. After washing 3 times with PBS (pH 7.2), add horseradish peroxidase (HRPO) -conjugated streptavidin, further incubate for 1 hour at 4 ° C, wash 3 times with PBS (pH 7.2), add peroxidase substrate and room temperature. The absorbance at 405 nm was measured with a microplate reader (Molecular Devices Corp., USA). Biotinylation of fucosylated fetuin was purified by Centricon-30 (Amicon Co., USA) after treatment with sulfo-NHS-biotin reagent (Pierce Co., USA). HRPO-coupled streptavidin, HRPO oxidation and NaBH_ThreeIt was prepared by conjugation of streptavidin by reductive amination method using CN. The measurement results are shown in Table 1.
[Table 1]

[0038]
Example 11
Of various glycan-linked liposomes by chloramine T method¹²⁵I sign
Chloramine T (Wako Pure Chemical Co., Japan) solution and sodium disulfite solution were prepared and used at 3 mg / ml and 5 mg / ml, respectively. Each of the 13 types of sugar chain-bound liposomes and tris (hydroxymethyl) aminomethane-bound liposomes prepared according to (Example 4) to (Example 9) was separately placed in an Eppendorf tube, 50 μl,¹²⁵15 μl of I-NaI (NEN Life Science Product, Inc. USA) and 10 μl of chloramine T solution were added and reacted. Every 5 minutes, 10 μl of chloramine T solution was added, and this operation was repeated twice. After 15 minutes, 100 μl of sodium disulfite was added as a reducing agent to stop the reaction. Next, it was placed on a Sephadex G-50 (Phramacia Biotech. Sweden) column chromatography, eluted with PBS, and the labeled product was purified. Finally, unlabeled-liposome complex was added to achieve a specific activity (4 x 10⁶ Bq / mg protein)¹²⁵An I-labeled liposome solution was obtained.
[0039]
Example 12
Measurement of various sugar chain-bound liposome complexes from the intestinal tract to the blood in mice
To male ddY mice (7 weeks old) fasted except for water all day and night, according to (Example 11)¹²⁵After forced administration into the intestinal tract with an oral sonde for mice, 0.2 ml of 13 types of I-labeled sugar chain linkages and tris (hydroxymethyl) aminomethane-linked liposome complex as a protein amount of 3 μg / unit, Ten minutes later, 1 ml of blood was collected from the lower aorta under Nembutal anesthesia. And in the blood¹²⁵I radioactivity was measured with a gamma counter (Alola ARC300). Furthermore, in order to investigate the in vivo stability of various liposome complexes, serum of each blood was rechromatographed with Sephadex G-50, and all of them showed high radioactivity in high molecular weight void fractions. The liposome complex was stable in vivo. The amount of radioactivity transferred from the intestinal tract into the blood was expressed as the ratio of radioactivity per ml of blood relative to the total radioactivity administered (% dose / ml blood). The results are shown in FIGS.
[0040]
【The invention's effect】
The above-mentioned sugar chain-modified liposome of the present invention is innovative in that it is excellent in intestinal absorbability and can be administered in a new dosage form that is not seen in conventional preparations using liposomes, through the intestinal tract. It is. Furthermore, intestinal absorbability can be controlled by setting the amount of sugar chain bound to the liposome and selecting the sugar chain, which enables efficient and safe side effects of drugs or genes via the intestine. It can be transferred to tissues and is particularly useful in the medical and pharmaceutical fields.
[Brief description of the drawings]
FIG. 1 is a schematic diagram showing a structural example of a liposome modified with a lactose disaccharide chain.
FIG. 2 is a schematic diagram showing an example of the structure of a liposome modified with a 2'-fucosyl lactose trisaccharide chain.
FIG. 3 is a schematic diagram showing a structure example of a liposome modified with a difucosyl lactose tetrasaccharide chain.
FIG. 4 is a schematic view showing a structural example of a liposome modified with 3-fucosyl lactose trisaccharide chain.
FIG. 5 is a schematic view of a liposome bound with tris (hydroxymethyl) aminomethane as a comparative sample.
FIG. 6 is a view showing the amount of transfer of three types of liposome complexes with different lactose disaccharide chain binding amounts into the blood 10 minutes after administration in the intestinal tract together with the control.
FIG. 7 is a view showing, together with controls, the amount of transfer of three types of liposome complexes in which the binding amount of 2′-fucosyl lactose trisaccharide chain is changed into the blood 10 minutes after intestinal administration.
FIG. 8 is a view showing, together with controls, the amount of three liposome complexes with different amounts of difucosyl lactose tetrasaccharide chains transferred into the blood 10 minutes after administration in the intestinal tract.
FIG. 9 is a view showing the amount of transfer of three types of liposome complexes with different binding amounts of 3-fucosyl lactose trisaccharide chain into the blood 10 minutes after administration in the intestinal tract together with the control.

Claims (7)

A liposome modified with a sugar chain selected from a lactose disaccharide chain , a 2′-fucosyl lactose trisaccharide chain , a difucosyl lactose tetrasaccharide chain and a 3-fucosyl lactose trisaccharide chain , wherein the sugar chain contains a linker protein; A liposome characterized by being bound to a liposome membrane via The liposome according to claim 1, wherein the liposome surface and / or the linker protein is made hydrophilic. The liposome according to claim 2, wherein the liposome surface and / or the linker protein is hydrophilized with tris (hydroxymethyl) aminomethane. The liposome according to any one of claims 1 to 3, wherein the linker protein is human serum albumin or bovine serum albumin . The liposome according to any one of claims 1 to 4, wherein the liposome has an intestinal absorption function. The liposome according to any one of claims 1 to 5 , wherein the amount of sugar chain binding on the liposome membrane is adjusted.   A liposome preparation comprising a drug or gene encapsulated in the liposome according to any one of claims 1 to 6.

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