JP4904573B2 - Composition for enhancing uptake of target substance into brain capillary endothelial cells - Google Patents

Description

  The present invention relates to a composition for enhancing uptake of a target substance into brain capillary endothelial cells.

  In recent years, development of vectors for reliably delivering target substances such as drugs and nucleic acids to target sites has been actively conducted. For example, in gene therapy, viral vectors such as retroviruses, adenoviruses, and adeno-associated viruses have been developed as vectors for introducing a target gene into target cells. However, since viral vectors have problems such as difficulty in mass production, antigenicity, and toxicity, liposome vectors with few such problems are attracting attention. Liposome vectors also have the advantage that directivity with respect to target sites can be improved by introducing functional molecules such as antibodies, proteins, and sugar chains on the surface thereof.

  For example, liposomes in which the liposome membrane surface is modified with a hydrophilic polymer (for example, polyalkylene glycol such as polyethylene glycol) have been developed (Patent Documents 1 to 4). According to this liposome, the directivity of the liposome with respect to tumor cells can be improved by improving the blood retention of the liposome.

  In addition, liposomes whose liposome membrane surface is modified with a maleimide group have been developed (Patent Documents 3 to 6). According to this liposome, it is possible to introduce various substances (for example, an antibody, a transferrin, a polyalkylene glycol, etc.) onto the surface of the liposome membrane by utilizing a reaction between a maleimide group and a thiol group.

Furthermore, as a non-viral vector that can be transferred from the blood into the brain, a liposome in which polyethylene glycol introduced on the surface of the liposome membrane is modified with the rat transferrin receptor antibody OX-26 (Non-patent Document 1) is introduced on the surface of the liposome membrane. Liposomes obtained by modifying polyethylene glycol with a human insulin receptor antibody (Non-patent Document 2) have been developed.
JP-A-1-249717 JP-A-2-149512 JP-A-4-346918 JP 2004-10482 A JP-A-11-106391 JP-A-2005-29683 Huwyler, J. et al., "Proceedings of the National Academy of Sciences of the United States of America", 1996, Vol. 93, p.14264-14169 Zhang, Y. et al., “Human Gene Therapy”, 2003, Vol. 14, p.1-12

The brain has a blood-brain barrier that is composed of brain capillary endothelial cells that are tightly bound together and covered with pericytes and astrocytes, so that the target substance can be delivered into the brain. Is not easy.
However, if the uptake of the target substance into brain capillary endothelial cells can be enhanced, it is considered that the delivery efficiency of the target substance in the brain can be improved.
Therefore, an object of the present invention is to provide a composition for enhancing the uptake of a target substance into brain capillary endothelial cells.

In order to solve the above problems, the present invention is a composition for enhancing the uptake of a target substance into brain capillary endothelial cells, having a hydrophilic polymer having a maleimide group introduced on the surface thereof, Provided is the composition containing a liposome in which a target substance is encapsulated.
In the composition of the present invention, the hydrophilic polymer is preferably ethylene glycol.

  Liposomes that have a hydrophilic polymer with a maleimide group introduced on the surface and encapsulate the target substance inside can efficiently migrate to the inside of the brain capillary endothelial cells. Incorporation into cells can be enhanced.

  As long as the liposome contained in the composition of the present invention is a closed vesicle having a lipid bilayer structure, it may be a multilamellar liposome (MLV), SUV (small unilamellar vesicle), or LUV (large unilamellar vesicle). ), Single membrane liposomes such as GUV (giant unilamellar vesicle).

  The size of the liposome contained in the composition of the present invention is not particularly limited, but is usually 50 to 500 nm in diameter, preferably 50 to 200 nm in diameter, and more preferably 50 to 80 nm in diameter.

  The liposome contained in the composition of the present invention has a hydrophilic polymer into which a maleimide group has been introduced (hereinafter referred to as “maleimide group-introduced hydrophilic polymer”) on the surface. Examples of the presence of the maleimide group-introduced hydrophilic polymer on the surface of the liposome include an embodiment in which the maleimide group-introduced hydrophilic polymer is exposed from the liposome membrane in a state of being bound to the liposome membrane constituent.

  In the maleimide group-introduced hydrophilic polymer, the maleimide group may be introduced at the end of the main chain of the hydrophilic polymer, or may be introduced at the end of the side chain, but is introduced at the end of the main chain. It is preferable. In the maleimide group-introduced hydrophilic polymer, the end of the main chain may be bonded to the liposome membrane component, or the end of the side chain may be bonded to the liposome membrane component. Is preferably bound to a liposome membrane constituent. That is, in the maleimide group-introduced hydrophilic polymer, it is preferable that the maleimide group is introduced into one end of the main chain of the hydrophilic polymer and the other end of the main chain of the hydrophilic polymer is bonded to the liposome membrane constituent.

  The liposome contained in the composition of the present invention is a hydrophilic polymer into which a maleimide group has not been introduced in addition to a liposome membrane constituent component bonded to a maleimide group-introduced hydrophilic polymer (hereinafter referred to as “maleimide group non-introduced hydrophilic polymer”). Liposome membrane constituents bound to.). When the liposome contains a liposome membrane component bonded to a maleimide group-non-introduced hydrophilic polymer, a liposome membrane component bonded to the maleimide group-introduced hydrophilic polymer, and a liposome membrane component bonded to a maleimide group-non-introduced hydrophilic polymer; By adjusting the content ratio, the in vivo retention of liposomes in blood can be adjusted.

  The content of the liposome membrane constituent component bound to the maleimide group-introduced hydrophilic polymer is not particularly limited, but is usually 0.1 to 10% (molar ratio), preferably 0.5, of the total amount of the liposome membrane constituent component. -5% (molar ratio), more preferably 0.5-1% (molar ratio). When the content of the liposome membrane component bound to the maleimide group-introduced hydrophilic polymer is within the above range, the liposome can efficiently migrate into the brain capillary endothelial cells.

  The content of the liposome membrane component bonded to the maleimide group non-introduced hydrophilic polymer is not particularly limited, but the liposome membrane component bonded to the maleimide group-introduced hydrophilic polymer and the maleimide group non-introduced hydrophilic polymer The total content of the bound liposome membrane constituents is usually 2 to 20% (molar ratio), preferably 3 to 10% (molar ratio), more preferably 5 to 10% (molar ratio) of the total amount of the liposome membrane constituents. Ratio). When the total content is in the above range, the in vivo retention of liposomes in blood can be effectively improved.

  The liposome membrane component is not particularly limited as long as it does not inhibit the formation of the lipid bilayer, and examples thereof include lipids, membrane stabilizers, antioxidants, and charged substances.

  Lipid is an essential component of the liposome membrane, and its content is usually 70 to 100% (molar ratio), preferably 80 to 100% (molar ratio), more preferably 90 to 100% (molar ratio) of the total amount of liposome membrane components. Molar ratio).

  Examples of lipids include phospholipids, glycolipids, sterols, saturated or unsaturated fatty acids, and the like.

  Examples of the phospholipid include phosphatidylcholine (e.g., dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine), phosphatidylglycerol (e.g., dioleoylphosphatidylglycerol, dilauroylphosphatidylglycerol, Dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, etc.), phosphatidylethanolamine (eg dioleoylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoy) Phosphatidylethanolamine, distearoyl phosphatidyl diethanolamine), phosphatidylserine, phosphatidylinositol, phosphatidic acid, cardiolipin, sphingomyelin, egg yolk lecithin, soybean lecithin, etc. These hydrogenated products thereof.

  Examples of glycolipids include glyceroglycolipid (eg, sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, glycosyl diglyceride), glycosphingolipid (eg, galactosyl cerebroside, lactosyl cerebroside, ganglioside) and the like. Can be mentioned.

  Examples of sterols include animal-derived sterols (for example, cholesterol, cholesterol succinic acid, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol), plant-derived sterols (phytosterol) (for example, stigmasterol, sitosterol, campesterol, Brush casterol), microorganism-derived sterol (for example, timosterol, ergosterol) and the like.

  Examples of saturated or unsaturated fatty acids include saturated or unsaturated fatty acids having 12 to 20 carbon atoms such as palmitic acid, oleic acid, stearic acid, arachidonic acid, and myristic acid.

  The membrane stabilizer is an optional component of the liposome membrane that can be added to physically or chemically stabilize the liposome membrane or to regulate the fluidity of the liposome membrane, and its content is a component of the liposome membrane constituent. The total amount is usually 0 to 30% (molar ratio), preferably 0 to 20% (molar ratio), and more preferably 0 to 10% (molar ratio).

  Examples of the film stabilizer include sterol, glycerin or fatty acid ester thereof. Specific examples of sterols are the same as those described above, and examples of glycerin fatty acid esters include triolein and trioctanoin.

  The antioxidant is an optional component of the liposome membrane that can be added to prevent the oxidation of the liposome membrane, and its content is usually 0-30% (molar ratio), preferably 0-20, of the total amount of the liposome membrane constituents. % (Molar ratio), more preferably 0 to 10% (molar ratio).

  Examples of the antioxidant include tocopherol, propyl gallate, ascorbyl palmitate, butylated hydroxytoluene and the like.

  The charged substance is an optional component of the liposome membrane that can be added to impart a positive charge or negative charge to the liposome membrane, and its content is usually 0 to 30% (molar ratio) of the total amount of the liposome membrane constituents, preferably It is 0 to 20% (molar ratio), more preferably 0 to 10% (molar ratio).

  Examples of the charged substance that imparts a positive charge include saturated or unsaturated aliphatic amines such as stearylamine and oleylamine; saturated or unsaturated cationic synthetic lipids such as dioleoyltrimethylammonium propane, and the like. Examples of the charged substance to be provided include dicetyl phosphate, cholesteryl hemisuccinate, phosphatidylserine, phosphatidylinositol, and phosphatidic acid.

  The liposome membrane constituent component to which the maleimide group-introduced hydrophilic polymer or the maleimide group-non-introduced hydrophilic polymer is bound is not particularly limited, but is preferably a lipid or a membrane stabilizer, such as phospholipid, sterol or fatty acid. More preferably it is. When the liposome membrane component to which the hydrophilic polymer with maleimide group introduced or the hydrophilic polymer with no maleimide group is bonded is a lipid or a membrane stabilizer, in particular phospholipid, sterol or fatty acid, the retention of posomes in the blood in vivo is improved. It can be improved effectively.

  The hydrophilic polymer is not particularly limited as long as it can improve the blood retention of liposomes in vivo, and examples thereof include polyalkylene glycols (for example, polyethylene glycol, polypropylene glycol, polytetramethylene glycol, polyhexamethylene). Methylene glycol, etc.), dextran, pullulan, ficoll, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer, amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin, carrageenan, etc. Is mentioned. Of these, polyalkylene glycol is preferable, and polyethylene glycol is more preferable.

  When the hydrophilic polymer is a polyalkylene glycol, the molecular weight is usually 500 to 5000, preferably 1000 to 3000, and more preferably 2000 to 3000. When the molecular weight of the polyalkylene glycol is in the above range, the in vivo retention of liposomes in blood can be effectively improved.

  A substituent such as an alkyl group, an alkoxy group, a hydroxyl group, a carbonyl group, an alkoxycarbonyl group, or a cyano group may be introduced into the hydrophilic polymer.

  Examples of the alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, s-butyl group, t-butyl group, n-pentyl group, isopentyl group, and t-pentyl. And a linear or branched alkyl group having 1 to 5 carbon atoms such as a group and a neopentyl group.

  Examples of the alkoxy group include straight-chain C1-C5 such as methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, s-butoxy group, and t-butoxy group. Or a branched alkoxy group is mentioned.

  The liposome membrane component and the hydrophilic polymer include a functional group possessed by the liposome membrane component (including a functional group artificially introduced into the liposome membrane component) and a functional group possessed by the hydrophilic polymer (hydrophilic polymer). And a functional group introduced artificially)) through a covalent bond formed by the reaction. Examples of combinations of functional groups that can form a covalent bond include amino groups / carboxyl groups, amino groups / acyl halide groups, amino groups / N-hydroxysuccinimide ester groups, amino groups / benzotriazole carbonate groups, amino groups / aldehydes. Group, thiol group / maleimide group, thiol group / vinylsulfone group, hydroxyl group / carboxyl group and the like, and the reaction between these functional groups can be performed according to a known method.

  The hydrophilic polymer and the maleimide group are a functional group possessed by the hydrophilic polymer (including a functional group artificially introduced into the hydrophilic polymer) and a functional group possessed by the maleimide group-containing compound (an artificially composed maleimide group-containing compound). Can be linked via a covalent bond formed by reaction with a chemically introduced functional group. Examples of combinations of functional groups that can form a covalent bond include amino groups / carboxyl groups, amino groups / acyl halide groups, amino groups / N-hydroxysuccinimide ester groups, amino groups / benzotriazole carbonate groups, amino groups / aldehydes. Group, thiol group / maleimide group, thiol group / vinylsulfone group, hydroxyl group / carboxyl group and the like, and the reaction between these functional groups can be performed according to a known method.

  When the hydrophilic polymer is polyethylene glycol, for example, a maleimide group can be introduced into polyethylene glycol by reacting polyethylene glycol and hydroxymethylmaleimide. This reaction can be performed according to a known method such as JP-A-2005-29683.

The maleimide group-introduced hydrophilic polymer has, for example, the following formula: P-X-M [wherein P represents a residue of the hydrophilic polymer, X represents a direct bond or a linking group, and M represents a maleimide group. ]. In addition, as a coupling group represented by X, for example, a divalent hydrocarbon group which may have a hetero atom; -O-; -S-; -NH-; -COO-; -SS-; -NHCO-; -NHCONH-; -SO- and the like. Examples of the hetero atom include a nitrogen atom, an oxygen atom, a halogen atom (a fluorine atom, a chlorine atom, a bromine atom, etc.), a sulfur atom, and the like. Examples of the divalent hydrocarbon group that may have a hetero atom include, for example, an alkylene group such as an ethylene group, a trimethylene group, and a propylene group; —SCH ₂ CH ₂ COO—; —OCH ₂ CH ₂ COO—; _{NHCH 2 CH 2 COO -; -} SCH 2 CH 2 COOCH 2 -; - SOCH 2 CH 2 COO- , and the like.

  Liposomes can be prepared, for example, using a known method such as a hydration method, an ultrasonic treatment method, an ethanol injection method, an ether injection method, a reverse phase evaporation method, a surfactant method, or a freezing / thawing method. Moreover, the liposome with a fixed particle size distribution can be obtained by passing the liposome through a filter having a predetermined pore size. Further, according to a known method, conversion from multilamellar liposomes to single membrane liposomes and conversion from single membrane liposomes to multilamellar liposomes can be performed.

  The target substance is encapsulated inside the liposome contained in the composition of the present invention.

  The target substance is not particularly limited, and examples thereof include drugs, nucleic acids, peptides, proteins, sugars, or complexes thereof, and can be appropriately selected depending on the purpose of diagnosis, treatment, and the like. Examples of drugs include brain tumor therapeutic agents, HIV therapeutic agents, central nervous system drugs (psycho-neurological agents, anti-parkinsonian agents, antipyretic analgesic / anti-inflammatory agents, hypnosis / sedation, anticonvulsants, central muscle relaxants, anti-dementia Drugs), cerebral circulation improving drugs, anticoagulants, cerebral protectants, hormones, antibiotics and the like. “Nucleic acid” includes analogs or derivatives thereof (eg, peptide nucleic acid (PNA), phosphorothioate DNA, etc.) in addition to DNA or RNA. The nucleic acid may be either single-stranded or double-stranded, and may be either linear or circular.

  When the target substance is water-soluble, it is possible to encapsulate the target substance in the aqueous phase inside the liposome by adding the target substance to an aqueous solvent used when hydrating the lipid membrane in the production of liposomes. it can. When the target substance is fat-soluble, the target substance can be encapsulated in the lipid bilayer of the liposome by adding the target substance to an organic solvent used in the production of the liposome.

  The composition of the present invention can enhance the uptake of a target substance to both brain capillary endothelial cells constituting a living organism and brain capillary endothelial cells not constituting a living organism. In addition, the composition of the present invention can enhance the uptake of a target substance to brain capillary endothelial cells derived from any species. Examples of biological species from which brain capillary endothelial cells are derived include mammals such as humans, monkeys, cows, sheep, goats, horses, pigs, rabbits, dogs, cats, rats, mice, and guinea pigs.

  Although the dosage form of the composition of this invention is not specifically limited, For example, the dispersion liquid of a liposome is mentioned. As the dispersion solvent, for example, a buffer solution such as physiological saline, phosphate buffer, citrate buffer, and acetate buffer can be used. For example, additives such as sugars, polyhydric alcohols, water-soluble polymers, nonionic surfactants, antioxidants, pH regulators, hydration accelerators may be added to the dispersion.

  Another dosage form of the composition of the present invention includes a dried product (eg, a lyophilized product, a spray-dried product, etc.) of a liposome dispersion. To the dried product, a buffer solution such as physiological saline, phosphate buffer solution, citrate buffer solution or acetate buffer solution can be added and used as a liposome dispersion.

  The administration route of the composition of the present invention includes, for example, parenteral administration such as intravenous, intraperitoneal, subcutaneous, nasal, etc. The dosage and the number of administrations are the kind of target substance enclosed in the liposome, It can be appropriately adjusted according to the amount and the like.

  Since the liposome contained in the composition of the present invention can efficiently migrate into the brain capillary endothelial cells, the target substance can be administered by encapsulating the target substance in the liposome and administering it to the target animal. Uptake into brain capillary endothelial cells can be enhanced.

[Example 1] Analysis of liposome pharmacokinetics (in vivo) Preparation of liposome (1) Preparation of liposome 1 (mal-PEG liposome) 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) (manufactured by NOF Corporation) bound to maleimide group-introduced polyethylene glycol, Egg yolk phosphatidylcholine (EPC) (manufactured by NOF Corporation), cholesterol (manufactured by Avanti) and DSPE-PEG2000 (manufactured by Avanti) dissolved in chloroform at 0.05: 7: 3: 0.5 (molar ratio) and mixed, eggplant type The solvent was evaporated to dryness in the flask to prepare a lipid membrane.

  The structural formula of DSPE bonded to maleimide group-introduced polyethylene glycol is as follows.

  The structural formula of DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000]) is as follows.

When preparing the lipid membrane, [ ³ H] CHE (manufactured by PerkinElmer Life Sciences Japan) was added as an RI tracer.
A PBS solution was poured onto the lipid membrane and applied to a bath-type ultrasonic cleaner to prepare liposomes. Subsequently, the size of the liposomes was adjusted to about 100 nm using Mini-Extruder (a polycarbonate membrane filter having a pore size of 0.1 μm, manufactured by Avanti). The size of the liposome was measured by a dynamic light scattering method using ELS-8000HO (manufactured by Otsuka Electronics Co., Ltd.).
Thus, a liposome having a hydrophilic polymer having a maleimide group introduced on its surface (PEG liposome) was prepared as the liposome 1.

(2) Preparation of liposome 2 (PEG liposome) Except that DSPE bound to maleimide group-introduced polyethylene glycol is not used, the same operation as in (1) is performed, and liposome 2 having polyethylene glycol on its surface (PEG) Liposome).

(3) Production of liposome 3 (Tf-PEG liposome) Liposome 3 was produced according to the literature (Int. J. Pharm. 281, 25-33, 2004.) of Hatakeyama, H. et al. That is, transferrin and 3- (2-pyridyldithio) propionic acid N-hydroxysuccinimide (SPDP) were mixed at 1: 1.5 (molar ratio) in phosphate buffered saline (PBS (-)) at room temperature. After reacting for 30 minutes, add DTT to a final concentration of 50 mM, react at room temperature for 30 minutes, and remove reaction byproducts using Sephadex G-25 Fine gel (Amersham Biosciences) The following formula: MPA-NH-Tf [wherein MPA represents a 3-mercaptopropionyl group, Tf represents a transferrin residue, and -NH- is derived from the amino group of transferrin. ] The transferrin derivative (I) represented by this was prepared. Next, transferrin derivative (I) was added to the solution of liposome 1 and stirred at 4 ° C. for O / N, so that transferrin derivative (I) was bound to the maleimide group introduced into polyethylene glycol via —S—. Thus, a liposome having transferrin-modified polyethylene glycol on its surface (Tf-PEG liposome) was prepared as liposome 3.

(4) Preparation of liposome 4 (Lf-PEG liposome) Except that lactoferrin was used instead of transferrin, the same operation as in (3) was performed, and liposome 4 having a lactoferrin-modified polyethylene glycol on its surface (Lf -PEG liposome).

(5) Preparation of liposome 5 (Av-PEG liposome) Except that avidin was used instead of transferrin, the same operation as in (3) was performed, and liposome 5 having an avidin-modified polyethylene glycol on its surface (Av -PEG liposome).

(6) Preparation of liposome 6 (DOTAP liposome)
1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-Dioleoyl-3-trimethyl-ammonium propane (DOTAP) were mixed at a molar ratio of 1: 1 and dissolved in chloroform. Was evaporated to prepare a lipid membrane. PBS was added to the lipid membrane and treated with a bath-type sonicator. Thus, a liposome containing DOTAP as a lipid membrane component (DOTAP liposome) was prepared as the liposome 6.

(7) Preparation of liposome 7 (DOTAP-R8 liposome) The same treatment as in (6) was carried out except that 20% (molar ratio) of stearylated octaarginine was contained in the lipid membrane. Liposomes containing DOTAP as a component and having octaarginine on the surface (DOTAP-R8 liposome) were prepared.

2. Administration to mice Each liposome was administered to ddY mice (male, 6-7 weeks old, manufactured by Japan SLC) at a concentration of 4 nmol lipid / g body weight via the tail vein. Blood, brain, heart, lung, liver, spleen, kidney, and skeletal muscle were taken out from 3 mice at a predetermined time after administration, and their weights were measured, respectively. Soluene-350 (manufactured by Perkin Elmer Life Sciences) In addition, it was dissolved by heating at 50 ° C. for 5 hours, Hionic Flour (manufactured by Perkin Elmer Life Sciences) was added, and the radioactivity was measured with a liquid scintillation counter.

  The blood concentration of liposomes was calculated as the radioactivity (% ID / mL) per dose in whole blood, and the blood retention of liposomes was examined based on this. The concentration of liposomes in each organ was calculated as the radioactivity (ID% / g tissue) per dose of each organ, and based on this, the transfer rate of liposomes to each organ was examined. In addition, what subtracted the contribution (blood concentration x blood vessel space) of the radioactivity derived from a blood component from the measured value of each organ was made into the radioactivity of each organ. In addition, the vascular space was calculated from the amount of PEG liposomes at an early time when it was assumed that there was no transfer to the organ.

The measurement result of the blood concentration of the liposome is shown in FIG.
As shown in FIG. 1, the blood concentration of liposome 1 (mal-PEG liposome) is lower than the blood concentration of liposome 2 (PEG liposome), but higher than the blood concentration of other liposomes. 1 (mal-PEG liposome) showed high blood retention.

The measurement results of the liposome concentration in each organ are shown in FIG. In FIG. 2, the second column from the left is the concentration of liposome 1 (mal-PEG liposome) in each organ.
As shown in FIG. 2, the rate of transfer of liposome 1 (mal-PEG liposome) to the liver and spleen, which are the major organs of the reticuloendothelial system, is low, and liposome 1 (mal-PEG liposome) is blood from the reticuloendothelial system. I was able to counter the exclusion from the inside.

The measurement result of the brain concentration of liposome is shown in FIG.
As shown in FIG. 3, the transfer rate of liposome 1 (mal-PEG liposome) to the brain exceeded 0.1%, which was significantly higher than the transfer rate of liposomes 2 to 7 to the brain (liposomes 3 and 6 were P <0.05, reliability of other liposomes is P <0.01).

[Example 2] Incorporation of liposomes into brain capillary endothelial cells (in vitro)
Liposomes were prepared in the same manner as in Example 1. At that time, 1% (molar ratio) of rhodamine-labeled lipid (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-lissamine rhodamine B sulfonyl: Rho-DOPE) was used. )added. Each liposome was added at a concentration of about 0.2 pmol lipid / cell in a DMEM culture solution of mouse brain capillary endothelial cells MBEC4 and incubated at 37 ° C. for 2 hours. The cell surface was washed with a colorless and transparent DMEM culture solution, and MBEC4 in the colorless and transparent DMEM culture solution was observed with a confocal laser scanning microscope LSM510 (manufactured by Carl Zeiss).

The observation result by a confocal laser scanning microscope is shown in FIG.
In FIG. 4, A (the upper left figure) shows the results when none of the liposomes is administered, and B (the upper middle figure) shows the results when the liposomes 2 (PEG liposomes) are administered. C (upper right figure) shows the results when liposome 1 (mal-PEG liposome) was administered.
When liposome 1 (mal-PEG liposome) was administered (upper right figure C), a clearer amount of rhodamine was observed than in other cases.

In FIG. 4, the left column (vertical 3 diagram) of D (bottom three columns) shows a cell cross-sectional view when none of the liposomes is administered, and the middle column of D (vertical 3 diagram). Shows a cell cross-sectional view when liposome 2 (PEG liposome) is administered, and the right column of D (longitudinal 3 diagram) shows a cell cross-sectional view when liposome 1 (mal-PEG liposome) is administered.
Liposome 1 (mal-PEG liposome) was not adsorbed on the cell surface but was taken into the cells. Liposome 1 (mal-PEG liposome) was accumulated around the nucleus.

It is a figure which shows the measurement result of blood concentration. It is a figure which shows the measurement result of the density | concentration in each organ of a liposome. It is a figure which shows the measurement result of the brain concentration of a liposome. It is a figure which shows the observation result by a confocal laser scanning microscope.

Claims (1)

A composition for enhancing the uptake of a target substance into brain capillary endothelial cells, comprising a polyethylene glycol having a maleimide group introduced on the surface thereof , wherein the target substance contains a liposome encapsulated therein Composition.