JP2010030915A - Composite of cationic polysaccharide and magnetic particle - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an MRI contrast medium stable in a culture solution and enabling unerring MRI diagnosis. <P>SOLUTION: The composite of a cationic polysaccharide derivative and a magnetic metal oxide has the following properties: the aggregation degree is ≤4.0 times when the composite stands still in D-MEM (Dulbecco's Modified Eagle Medium) containing 20% fetal bovine serum at 37°C under 5% CO<SB>2</SB>condition for 24hr. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a complex of a polysaccharide derivative and a magnetic metal oxide (also referred to herein as “polysaccharide magnetic particle complex”). The present invention relates to a polysaccharide magnetic particle composite which is particularly suitable for magnetic labeling of cells and tissues and further suitable for MRI diagnosis.

In the transplantation of cells and tissues today, the evaluation of the transplanted cells and tissues is generally performed by a method of observing a life phenomenon that is expressed when the cells or tissues function normally. For example, in islet cell transplantation for patients with congenital type 1 diabetes, colonization of transplanted cells is indirectly evaluated by measuring portal blood pressure and insulin secretion after transplantation. However, it is not easy to appropriately and early diagnose the state of cells and tissues immediately after transplantation by such an indirect evaluation method. For this reason, there are cases where treatment for appropriately maintaining transplanted cells and tissues is performed in an excessively small amount or excessively, and the clinical risks and costs caused thereby cannot be overlooked.
On the other hand, even when cells and tissues are transplanted properly, rapid cell death due to immune rejection after transplantation, cell necrosis due to damage at the time of tissue extraction, etc. may be seen, and postoperatively There is a great demand for accurate follow-up.

Against this background, in recent years, cells and tissues are magnetically labeled using a magnetic metal oxide such as superparamagnetic iron oxide (SPIO) before transplantation, and nuclear magnetic resonance imaging (MRI) is performed after transplantation of cells and tissues. It has been devised to perform follow-up observations using a computer.
For example, it has been proposed to perform cell labeling using a complex of a magnetic metal oxide and a polysaccharide such as dextran (Patent Document 1, Patent Document 2, Patent Document 3, and Patent Document 4). Such a polysaccharide magnetic particle complex has advantages such as low toxicity and slow blood clearance, but has a negative surface charge and low labeling ability for general transplanted cells such as islet cells. There was a problem that it was difficult to detect by MRI after transplantation.
In addition, it is known that the iron oxide complex of reduced polysaccharide is stable at the temperature required for sterilization (patent document 5).

On the other hand, a complex of a polysaccharide having a positive surface charge and a magnetic metal oxide has also been proposed. For example, magnetic particles in which a magnetic metal oxide is coated with diethylaminodextran have been reported (Patent Document 6, Patent Document 7, and Non-Patent Document 1). The cell labeling ability of these complexes was improved as compared with that of the previous complex, but no MRI images in vivo have been reported.
. In addition, iron oxide nanoparticles (ION) produced by adding ammonia water to a mixed aqueous solution of Fe ²⁺ and Fe ³⁺ ions containing pullulan and its derivatives and coprecipitating them are rat bone marrow mesenchymal stem cells (MSC). It has been reported that the MRI contrast ability was shown (Non-patent Document 2).

Japanese Patent No. 2921984 Japanese Patent No. 2939336 Japanese Patent No. 2726520 Japanese Patent No. 4070150 JP-T-2002-541218 JP-T-9-501675 JP 2007-112904 A C. Chouly et.al., Journal Microencapsulation, 1996, 13, 3, 245-255 Junichiro Shiro et al., Regenerative Medicine (Journal of Japanese Society for Regenerative Medicine), VOL. 7, Suppl., 290, 2008

  The present invention provides a polysaccharide magnetic particle composite that is superior in cell labeling ability compared to conventional polysaccharide magnetic particle composites. More specifically, the present invention is stable in a culture solution and enables accurate MRI diagnosis. It is an object to provide an MRI contrast agent.

The present inventors considered that the conventional polysaccharide magnetic particle complex causes aggregation in the culture medium, and this may prevent stable cell labeling. Thus, as a result of examining the production method and physical properties of various polysaccharide magnetic particle composites, the present inventors have succeeded in obtaining a polysaccharide magnetic particle composite satisfying a specific degree of aggregation. And it discovered that this polysaccharide particle complex had a stable cell labeling ability and therefore an excellent MRI imaging ability, and completed the present invention.
Furthermore, the present inventors have found that a complex obtained by using a cationic polysaccharide derivative in which the terminal aldehyde (reducing end) of the polysaccharide derivative is carboxylated or alcoholated is difficult to aggregate in the culture solution, The present invention has been completed.
That is, the present invention is as follows.

(1) a complex of a cationic polysaccharide derivative and a magnetic metal oxide having the following properties:
In D-MEM containing 20% fetal bovine serum, the degree of aggregation when left at 37 ° C. and 5% CO ₂ for 24 hours is 4.0 times or less (hereinafter referred to as “the composite of the present invention”). Also called "body.") (2) The complex according to (1), wherein the terminal aldehyde of the cationic polysaccharide derivative is carboxylated or alcoholated.
(3) The composite according to (1) or (2), obtained by coating a magnetic metal oxide with a cationic polysaccharide derivative and then heating at 50 to 120 ° C. for 5 minutes to 6 hours.
(4) The composite according to any one of (1) to (3), wherein the magnetic metal oxide is ferrite.
(5) The complex according to any one of (1) to (4), wherein the cationic polysaccharide derivative is an aminoalkyl etherified polysaccharide.
(6) The complex according to any one of (1) to (5), wherein the polysaccharide is at least one selected from dextran, dextrin, cellulose, agarose, starch, and pullulan. (7) The complex according to any one of (1) to (6), wherein the degree of substitution of the cationic polysaccharide derivative is within the range of 0.2 to 0.6 mol / AGU.
(8) The composite according to any one of (1) to (7), having the following properties:
When allowed to stand at 37 ° C. and 5% CO ₂ for 1 hour in D-MEM containing 20% fetal calf serum containing islet cells and a complex sufficient for the cells, the complex The labeled amount per 1 × 10 ⁶ cells is 0.6 μg or more in terms of the metal mass of the magnetic metal oxide.
(9) The complex according to any one of (1) to (8), comprising the cationic polysaccharide derivative within a range of 0.05 to 20 parts by mass per 1 part by mass of the metal in the magnetic metal oxide.
(10) The composite according to any one of (1) to (9), wherein the average primary particle diameter measured by a dynamic light scattering method is in the range of 5 to 100 nm.
(11) The complex according to any one of (1) to (10), wherein the T ₂ relaxation ability in the form of a water sol is in the range of 90 to 1000 (mM · sec) ⁻¹ .
(12) The complex according to any one of (1) to (11), which is a labeling agent for cells or tissues.
(13) The complex according to (12), wherein the cell or tissue is an islet cell or an islet.
(14) The complex according to (12) or (13), which is an MRI contrast agent.
(15) After coating the magnetic metal oxide with the cationic polysaccharide derivative, at 50 to 120 ° C. for 5 minutes to
The manufacturing method of the composite_body | complex in any one of (3)-(14) including heating for 6 hours.
(16) The method for producing a cationic polysaccharide derivative according to (15), wherein a terminal aldehyde of the polysaccharide is carboxylated or alcoholized.
(17) A cell labeling method comprising culturing cells in the presence of the complex according to any one of (1) to (14).

  The complex of the present invention does not aggregate for a long time even in a culture solution and is stable. Therefore, cells labeled with the complex of the present invention have a stable MRI imaging ability. In addition, since the complex of the present invention has low cytotoxicity, it is suitable as an MRI contrast agent for transplanted cells.

Hereinafter, the composite of the present invention will be described in more detail.
The complex of the present invention is a complex of a cationic polysaccharide derivative and a magnetic metal oxide, and has the following properties.
Aggregation degree is less than 4.0 times when left in D-MEM (Dulbecco's modified Eagle medium) containing 20% fetal bovine serum (FBS) at 37 ° C. and 5% CO ₂ for 24 hours. is there.

“D-MEM” is D-MEM (Dulbecco's Modified Eagle Medium) product number 11995-065 manufactured by Gibco BRL.
“%” Percent refers to volume%.
The “aggregation degree” is a value indicating the degree of increase in the size of the complex due to aggregation, and is obtained by dividing the average size of the aggregate after standing by the average primary particle diameter.
Here, the “average primary particle diameter” is the average diameter of individual particles (primary particles) of the complex of the cationic polysaccharide derivative and the magnetic metal oxide. The “average size of agglomerates” is the average size of all composites including the primary particles and agglomerates formed by agglomeration of the primary particles.
Here, the average primary particle diameter of the composite and the average size of the aggregate are values measured by a dynamic light scattering method (see, for example, Polymer J., 13, 1037-1043 (1981)). Specific sample preparation methods and measurement conditions are as follows.

(Sample preparation for measurement of average primary particle size)
The composite is diluted with a borate buffer having a pH of 9.0 to give a concentration of 0.05 mg / mL in terms of the mass of the metal in the metal oxide to obtain an aqueous sol of the composite.
(Sample preparation in measuring the average size of agglomerates)
The complex is diluted with D-MEM containing 20% of the fetal calf serum so as to have a concentration of 0.10 mg / mL in terms of the metal mass of the metal oxide, and is allowed to stand still in a 37 ° C. incubator for 24 hours. And then diluted to 1/2 with distilled water to obtain an aqueous sol.
(Measurement condition)
Device name: Autosizer 4700 (Malvern), temperature: 25 ° C., measurement angle: 90 ° C., wavelength: 514 nm

  The aggregation degree of the composite of the present invention is preferably 3.0 times or less, more preferably 2.0 times or less, further preferably 1.5 times, and further preferably 1.0 times or less.

The cationic polysaccharide derivative constituting the complex of the present invention is a polysaccharide having a cationic substituent (hereinafter also referred to as “cation group”) and having a positive charge as a whole.
Examples of the cationic group include a group containing a primary to quaternary amine. Preferably, it is a group containing a quaternary amine. Examples of such cationic groups include aminoaminoalkyl ether groups and aminosilane groups, with aminoalkyl ether groups being preferred. In the present invention, a polysaccharide having an aminoalkyl ether group is referred to as “aminoalkyl etherified polysaccharide”.
Aminoalkyl etherified polysaccharides can be produced using known methods.
For example, according to the method described in Chemistry and Industry, 1959, (11), 1490-1491, Japanese Patent Publication No. 59-30161, etc., for example, after adding an alkali such as NaOH to an aqueous solution or suspension of polysaccharide, amino The reaction can be carried out by adding an alkyl halide or a corresponding epoxide or an ammonioalkyl halide or a corresponding epoxide.

Neutral polysaccharides are preferable as the raw material polysaccharide. For example, glucose polymers such as dextran, dextrin, starch, glycogen, cellulose, curdlan, schizophyllan, lentinan, pestarothian, pullulan and the like; fructose polymers such as inulin and levan; mannose polymer Examples include mannan and the like; galactose polymers such as agarose and galactan; xylose polymers such as xylan and L-arabinose polymers such as arabinan, among which dextran, starch and cellulose are preferable, and dextran is particularly preferable. The weight average molecular weight of the polysaccharide is usually 3000 to 100,000, preferably 3000 to 70,000. Further, it is preferable to use a polysaccharide in which the terminal aldehyde is carboxylated (oxidized polysaccharide) or a polysaccharide in which the terminal aldehyde is alcoholated (reduced polysaccharide). Carboxylation of the terminal aldehyde can be performed by oxidizing the terminal aldehyde by an alkali treatment method or the like. The terminal aldehyde is alcoholated by reducing the terminal aldehyde by a method using sodium amalgam, a method using hydrogen gas in the presence of palladium carbon, a method using sodium borohydride (NaBH ₄ ), or the like. Can do.

  The aminoalkyl etherification of the polysaccharide can be carried out using an aminoalkyl etherifying agent such as an aminoalkyl halide or its corresponding epoxide, or an unsubstituted or substituted ammonioalkyl halide or its corresponding epoxide.

  The aminoalkyl halide or its corresponding epoxide is represented by the following formula (I).

In formula (I),
A ₁ represents an alkylene group,
R ¹ and R ² each independently represent a hydrogen atom or a hydrocarbon group, or represent a nitrogen-containing heterocycle formed together with the nitrogen atom to which R ¹ and R ² are bonded,
Y represents a halogen atom or an epoxy group (the following formula).

The alkylene group represented by A ₁ is preferably an alkylene group having 1 to 10 carbon atoms, more preferably 1 to 3 carbon atoms.

When at least one of R ¹ and R ² represents a hydrocarbon group, the hydrocarbon group may be saturated or unsaturated, chained or cyclic. The hydrocarbon group is, for example, an alkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, a cycloalkylalkyl group, a cycloalkenylalkyl group, an aryl group, or an aralkyl group. The hydrocarbon group is preferably an alkyl group having 1 to 3 carbon atoms.
When R ¹ and R ² form a nitrogen-containing heterocycle together with the nitrogen atom to which they are bonded, examples of the nitrogen-containing heterocycle include aziridine, pyrrolidine, pyrroline, pyrrole, piperidine, morpholine. , Indole, indoline, and isoindoline. The nitrogen-containing heterocycle is preferably a 5-membered ring or a 6-membered ring, and examples thereof include pyrrolidine, pyrroline, piperidine, and morpholine. Of these, pyrrolidine or piperidine is preferable.

  Specific examples of aminoalkyl halides or their corresponding epoxides include, for example, aminomethyl chloride, aminomethyl bromide, aminoethyl chloride, aminopropyl bromide, methylaminomethyl chloride, methylaminomethyl bromide, ethylaminoethyl chloride, ethyl Aminoethyl bromide, ethylaminopropyl chloride, propylaminopropyl chloride, dimethylaminomethyl chloride, dimethylaminoethyl chloride, diethylaminomethyl chloride, diethylaminoethyl chloride, diethylaminoethyl bromide, diethylaminopropyl chloride, dipropylaminoethyl bromide, dipropylaminopropyl Chloride, 1-pyrrolidinylmethyl chloride, -(1-pyrrolidinyl) ethyl chloride, 3- (1-pyrrolidinyl) propyl chloride, 1-piperidinylmethyl chloride, 2- (1-piperidinyl) ethyl chloride, 3- (1-piperidinyl) propyl chloride, or the like Of the corresponding epoxides.

  An unsubstituted or substituted ammonioalkyl halide or its corresponding epoxide is represented by the following formula (II).

In formula (II),
A ₂ represents an alkylene group,
R ³ , R ⁴ and R ⁵ each independently represent a hydrogen atom or a hydrocarbon group, or at least two of R ³ , R ⁴ and R ⁵ are combined with the nitrogen atom to which they are bonded. Represents a nitrogen-containing heterocycle formed by
Y represents a halogen atom or an epoxy group (following formula),
Z represents an anion.

The alkylene group represented by A ₂ is preferably an alkylene group having 1 to 10 carbon atoms, more preferably 1 to 3 carbon atoms.

When at least one of R ³ , R ⁴ and R ⁵ represents a hydrocarbon group, the hydrocarbon group may be saturated or unsaturated, and may be a chain or a ring Good. The hydrocarbon group is, for example, an alkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, a cycloalkylalkyl group, a cycloalkenylalkyl group, an aryl group, or an aralkyl group. The hydrocarbon group is preferably an alkyl group of 1 to 3.
When at least two of R ³ , R ⁴ and R ⁵ together with the nitrogen atom to which they are bonded form a nitrogen-containing heterocycle, examples of the nitrogen-containing heterocycle include aziridine and pyrrolidine. , Pyrroline, pyrrole, piperidine, morpholine, indole, indoline, isoindoline. The nitrogen-containing heterocycle is preferably a 5-membered ring or a 6-membered ring, and examples thereof include pyrrolidine, pyrroline, piperidine, and morpholine.
Examples of the anion of Z include halogen ions such as chlorine ion, fluorine ion and bromine ion, inorganic acid ions such as nitrate ion, and organic acid ions such as formate ion and acetate ion.

  Specific examples of the unsubstituted or substituted ammonioalkyl halide or the corresponding epoxide thereof include, for example (where the anion of Z is omitted), 2-chloroethyltrimethylammonium, 2-chloroethyltriethyl Ammonium, 2-chloroethyltripropylammonium, 2-chloroethyltrin-butylammonium, 3-chloropropyltrimethylammonium, 3-chloropropyltriethylammonium, 3-chloropropyltripropylammonium, 3-chloropropyltrin-butyl Ammonium, 3-chloro-2-hydroxypropyltrimethylammonium, 3-chloro-2-hydroxypropyltriethylammonium, 3-chloro-2-hydroxypropyltri-n-butylammonium 3-chloro-2-hydroxypropyltriiso-butylammonium, 3-bromo-2-hydroxypropyltrimethylammonium, 3-bromo-2-hydroxypropyltriethylammonium, 3-bromo-2-hydroxypropyltri-n-butylammonium , 3-bromo-2-hydroxypropyltriiso-butylammonium, or their corresponding epoxides.

  By performing aminoalkyl etherification of a polysaccharide using the aminoalkyl etherifying agent represented by the above formula (I) or (II), the hydroxy group of the polysaccharide is represented by the following formula (III) or (IV). The compound substituted with the group to be obtained is obtained.

In formulas (III) and (IV),
A ₃ and A ₄ each represents an alkylene group which may be substituted with a hydroxy group,
R ¹ , R ² , R ³ , R ⁴ , R ⁵ and Z are as defined above.
The alkylene group represented by A ₃ and A ₄ is preferably an alkylene group having 1 to 3 carbon atoms.
The preferred ranges of R ¹ , R ² , R ³ , R ⁴ , R ⁵ and Z are also as described above.

Specific examples of the aminoalkyl ether group include a dimethylaminomethyl ether group, a diethylaminoethyl ether group, a dipropylaminopropyl ether group, a diethylaminopropyl ether group, a 2- (1-pyrrolidinyl) ethyl ether group, and a trimethylammonio group. Ethyl ether group, triethylammonioethyl ether group, tripropylammonioethyl ether group, trimethylammoniopropyl ether group, triethylammoniopropyl ether group, trimethylammonio-2-hydroxypropyl ether group, triethylammonio-2- A hydroxypropyl ether group is mentioned.
In particular, diethylaminoethyl ether group, dimethylaminomethyl ether group, dipropylaminopropyl ether group, trimethylammonio-2-hydroxypropyl ether group, triethylammonio-2-hydroxypropyl ether group, tripropylammonio-2-hydroxy Propyl ether groups are preferred.

The amino group of the aminoalkyl ether group represented by the formula (III) can exist in the form of a salt. Examples of inorganic acid salts include hydrochloride, hydrofluoride, hydrobromide, and nitrate; examples of organic acid salts include formate and acetate.
The aminoalkyl etherified polysaccharide having an aminoalkyl ether group as represented by the formula (IV) is produced using the aminoalkyl etherifying agent represented by the formula (II) as described above. However, after substitution with the aminoalkyl ether group represented by the formula (III) using the aminoalkyl etherifying agent represented by the formula (I), the amino group of the aminoalkyl ether group is For example, it can be produced by converting it to an ammonium salt by reacting with an unsubstituted or substituted alkyl halide.

  In the cationic polysaccharide derivative used in the complex of the present invention, the terminal aldehyde is preferably carboxylated or alcoholated. By using such a cationic polysaccharide derivative, the degree of aggregation of 4.0 times or less can be achieved. Such a cationic polysaccharide derivative is, for example, as described above, as a raw material polysaccharide for producing it, a polysaccharide in which a terminal aldehyde is carboxylated (oxidized polysaccharide) or a polysaccharide in which a terminal aldehyde is alcoholized (reduced polysaccharide). Can be obtained.

The cationic polysaccharide derivative used in the complex of the present invention is preferably water-soluble.
The degree of substitution of the cationic polysaccharide derivative is preferably in the range of 0.2 to 0.6 mol / AGU, more preferably about 0.2 to 0.4 mol / AGU. AGU indicates anhydroglucose unit. When the complex of the present invention is used as a cell labeling agent, a high cell labeling amount can be obtained by setting the substitution degree within such a range.
When the cationic group is the aminoalkyl ether group, the degree of substitution is measured as follows.

(Measurement of substitution degree of aminoalkyl ether group)
The nitrogen content is measured according to the method described in Japanese Pharmacopoeia (Twelfth Amendment, 1991), General Test Method, Item 30, Nitrogen Determination Method, and the degree of substitution of aminoalkyl ether groups is calculated.

  In addition, a part of the hydroxyl group of the cationic polysaccharide derivative may be substituted with an anionic substituent (anionic group) or a nonionic substituent (nonionic group) as long as the cationicity of the polysaccharide derivative is not impaired. Good. Examples of the anionic group include a carboxyl group, a sulfate group, and a phosphate group. Examples of the nonionic group include a methoxyl group and an ethoxyl group.

The magnetic metal oxide constituting the composite of the present invention is a ferromagnetic particle.
Examples of such a magnetic metal oxide include a compound represented by the following formula (V).
(M ^II O) _l・ M ₂ ^III O ₃ (V)
In formula (V),
M ^II represents a divalent metal atom,
M ^III represents a trivalent metal atom,
l represents a real number within the range of 0-1.

In the above formula (V), the divalent metal atom M ^II, for example, magnesium, calcium, manganese, iron, nickel, cobalt, copper, zinc, strontium, barium, and the like. These may be used alone Or two or more of them can be used in combination. Examples of the trivalent metal atom ^MIII include aluminum, iron, yttrium, neodymium, samarium, europium, gadolinium, and the like. These may be used alone or in combination of two or more. it can.

Among the compounds represented by the above formula (V), the magnetic metal oxide is preferably a magnetic metal oxide in which M ^III is trivalent iron, that is, a ferrite represented by the following formula.
(M ^II O) _m · Fe ₂ O ₃ (V-1)
In formula (V),
M ^II represents a divalent metal atom,
m is a real number within the range of 0-1.

Here, examples of M ^II include the same metal atoms as mentioned in the formula (V). In particular, the magnetic metal oxide represented by the above formula (V-1) when M ^II is divalent iron, that is, the magnetic iron oxide represented by the following formula (V-2) is preferable.
_{(FeO) n · Fe 2 O} 3 (V-2)
In formula (V-2), n is a real number within the range of 0-1.

In the above formula (V-2), when n = 0, it is γ-iron oxide (γ-Fe ₂ O ₃ ), and when n = 1, it is magnetite (Fe ₃ O ₄ ). The magnetic metal oxide includes a magnetic metal oxide having crystal water.

The composite of the present invention preferably has a small coercive force and is superparamagnetic. In general, the magnetization of a magnetic material decreases as the particle diameter decreases, and this tendency becomes stronger when the particle diameter becomes 10 nm or less. Further, the coercive force of the magnetic material also decreases as the particle diameter decreases.
Therefore, the particle diameter of the magnetic metal oxide is preferably in the range of 2 to 20 nm, more preferably 3 to 15 nm, and further preferably 3 to 10 nm.

The complex of the present invention can be produced, for example, by a method using a cationic polysaccharide derivative in which a terminal aldehyde is carboxylated or alcoholated. Conditions other than this may be known conditions. For example, it can be produced as follows.
In the presence of a cationic polysaccharide derivative in an aqueous system, a mixed metal salt aqueous solution of a divalent metal salt and a trivalent metal salt is mixed with a basic aqueous solution to obtain the complex of the present invention in one step. Examples of the metal salt include a salt with one kind selected from mineral acids such as hydrochloric acid, sulfuric acid, and nitric acid. Usually, a salt with hydrochloric acid is preferred. The addition order of each substance is not particularly limited.

For preparing the above mixed metal salt aqueous solution, for example, when M ^II in the formula (V) is divalent iron and M ^III is trivalent iron, ferrous salt and ferric iron are used. The salt is preferably dissolved in the aqueous solvent at a ratio of about 1: 4 to about 3: 1 (molar ratio), more preferably about 1: 3 to about 1: 1 (molar ratio). In this case, a part of the ferrous salt, for example, about half of the other divalent metal salt, for example, magnesium, calcium, manganese, iron, nickel, cobalt, copper, zinc, strontium, barium, etc. is used. It can be replaced with a metal salt. The metal salt concentration of the mixed metal salt aqueous solution is not particularly limited, but is usually 0.1 to 5M, preferably 0.5 to 3M.

  Examples of the base contained in the basic aqueous solution include at least one selected from alkali metal hydroxides such as NaOH and KOH; ammonia; amines such as trimethylamine and triethylamine. It is usually preferred to use NaOH. The concentration of the basic aqueous solution can also be varied over a wide range, but is usually in the range of about 0.1 to 10N, preferably about 1 to 5N. The mixing ratio of the mixed metal salt aqueous solution and the basic aqueous solution is preferably such that the pH of the mixed reaction solution of the mixed metal salt aqueous solution and the basic aqueous solution is approximately neutral to pH 12, that is, the ratio of the metal salt to the base. Is about 1: 1 to about 1: 1.4 (specified ratio).

On the other hand, the amount of the cationic polysaccharide derivative can be preferably about 1 to 40 times based on the mass of the metal in the metal salt used. Further, the concentration of the aqueous solution of the cationic polysaccharide derivative is not strictly limited, but is usually about 1 to 70 w / v%, preferably about 5 to 60 w / v%. Addition and mixing of each aqueous solution can be carried out under stirring, usually without heating or heating at about 0 to 100 ° C., preferably about 20 to 80 ° C. If necessary, the pH is adjusted by adding a base or an acid. .
As a result, the magnetic metal oxide is coated with the cationic polysaccharide derivative.

After the magnetic metal oxide is coated with the cationic polysaccharide derivative as described above, the obtained reaction solution is preferably heated. The heating is performed at a temperature of 50 to 120 ° C., preferably 70 to 110 ° C., more preferably 90 to 105 ° C., for 5 minutes to 6 hours, preferably 30 minutes to 3 hours, more preferably 1 to 2 hours. By passing through this step, the T ₂ relaxation ability of the composite is improved.

The above mixing reaction and heating can be performed in an air atmosphere, but if desired, under an inert gas such as N ₂ and Ar gas, a reducing gas such as H ₂ gas, or an oxidizing gas such as O ₂ gas. Can also be done.
The reaction solution thus obtained can be purified and, if desired, adjusted to pH, concentrated, filtered, and dried.

The ratio of the cationic polysaccharide derivative to the magnetic metal oxide in the complex of the present invention can be varied within a wide range depending on the particle diameter of the magnetic metal oxide and the molecular weight of the cationic polyvalent derivative. Usually, the cationic polysaccharide derivative is 0.05 to 20 parts by weight, preferably 0.2 to 10 parts by weight, more preferably 0.5 to 5 parts by weight, based on 1 part by weight of the metal in the magnetic metal oxide. Preferably it contains 1-3 mass parts.
Here, the mass of the metal in the magnetic metal oxide is measured by atomic absorption spectrophotometry. That is, hydrochloric acid is added to the complex in the presence of a small amount of water, and the contained metal is completely decomposed to chloride, then diluted appropriately, and the absorbance at a specific wavelength between each metal reference solution is obtained. In comparison, the metal content is determined.
In addition, the content of the cationic polysaccharide derivative in the complex was determined according to Analytical Chem. , 25, 1656 (1953) and measured by the sulfuric acid-anthrone method. That is, a sulfuric acid-anthrone test solution is added to a solution obtained by appropriately diluting the composite sol to cause color development, and the absorbance is measured. At the same time, using the cationic polysaccharide derivative used in the production of the complex as a reference substance, the color is developed in the same manner, the absorbance is measured, and the content of the cationic polysaccharide derivative in the complex is determined from the ratio of the absorbances of the two.

  The average primary particle size of the composite of the present invention is preferably in the range of 5 to 100 nm, more preferably 10 to 75 nm. The definition of the average primary particle diameter is as described above. Moreover, the measuring method and measuring conditions of the average primary particle diameter are also as described above.

  The complex of the present invention is not a simple mixture but a compound of a magnetic metal oxide and a cationic polysaccharide derivative. This is because, for example, when a poor solvent is added after the reaction, the complex of the present invention is preferentially precipitated, and when the aqueous sol of the complex of the present invention is fractionated, a complex containing sugar and metal It can be understood from the fact that it can be separated into free polysaccharide derivatives.

In addition, the method of carboxylating or alcoholating the terminal aldehyde of the cationic polysaccharide derivative can be used as a method for improving the stability of the cationic polysaccharide magnetic particle composite or a method for producing a stable cationic polysaccharide magnetic particle composite. In addition, the method of heating the magnetic metal oxide with a polysaccharide derivative and then heating at 50 to 120 ° C. for 5 minutes to 6 hours is a method for increasing the T ₂ relaxation ability of the cationic polysaccharide magnetic particle composite, and a high T ₂ relaxation ability. It can be used as a method for producing a cationic polysaccharide magnetic particle composite having The preferable temperature and heating time in this case are the same as described above.

Moreover, it is preferable that the composite_body | complex of this invention has the following property.
When allowed to stand at 37 ° C. and 5% CO ₂ for 1 hour in D-MEM containing 20% fetal calf serum containing islet cells and a complex sufficient for the cells, the complex The labeling amount per 1 × 10 ⁶ cells is 0.6 μg or more, preferably 0.8 μg or more, more preferably 1.0 μg or more in terms of the metal mass of the magnetic metal oxide.
Here, “islet cells” are derived from mammals, preferably from mice or rats. Islet cells can be isolated from mammalian islets. For example, Joo Ho Tai et al., Diabetes, Vol. 55, November 2006 can be referred to as the separation method. In addition, the mouse pancreatic islet-derived cancer cell line “MIN6” (Ishihara H et al., Diabetologia. 1993 Nov; 36 (11): 1139-45, Jun-ichi Miyazaki et al., Endocrinology, Vol. 127, No. 1, 126-132 etc.) can be used as islet cells.
“Containing sufficient amount of the complex for the cells” means that 2 × 10 ⁶ cells in 3 ml of D-MEM containing 20% of the fetal bovine serum are contained in a magnetic metal oxide. It refers to the condition in which 300 μg of the complex exists in terms of metal mass.

The T ₂ relaxation capacity of the complex of the present invention in the form of water sol is preferably 90 to 1000 (mM).
• sec) ⁻¹ , more preferably in the range of 90 to 300 (mM · sec) ⁻¹ . Here, the “water sol” is a sol produced using distilled water as a solvent.
“T ₂ relaxation capability” is a measurement of T ₂ relaxation time by pulsed NMR at 20 MHz (magnetic field is about 0.5 Tesla) for water sol containing various concentrations of the complex of the present invention and solvent distilled water. Plot the relationship between the reciprocal of the obtained T ₂ relaxation time, 1 / T ₂ (unit: 1 / sec), and the metal concentration (unit: mM) in the measurement sample on the graph, and use the least square method. (Unit: 1 / mM · sec). Such range T ₂ relaxation ability can be obtained, for example, by coating a magnetic metal oxide with a cationic polysaccharide derivative and then heating at 50 to 120 ° C. for 5 minutes to 6 hours.

  The composite of the present invention can be used in industrial fields such as mechanical seal materials, magnetic clutches, and magnetic inks in the form of an aqueous sol. Used in preparations. This is because the complex of the present invention does not aggregate in the culture solution and thus has advantages such as high uptake efficiency into cells and low cytotoxicity. Among them, it is preferable to use as a labeling agent for cells or tissues for transplantation, particularly as a labeling agent for islet cells or islets for transplantation. The complex of the present invention is particularly preferably an MRI contrast agent.

When the complex of the present invention is used as a labeling agent for cells or tissues for transplantation, the complex is usually in the form of an aqueous sol. At this time, the concentration of the composite can be varied over a wide range, but is usually 0.1 mmol / L to 1 mol / L, preferably 1 to 5 mmol / L in terms of the amount of metal in the magnetic metal oxide. Is within the range. In preparing the aqueous sol, a medium such as D-MEM can be used as a solvent, and the medium may contain serum. For example, fetal bovine serum (FBS) may be contained in an amount of 40% by volume or less, preferably 20% by volume or less. By using such a culture solution as a solvent, the efficiency of uptake of the complex into cells, particularly pancreatic cells or tissues, is improved. Moreover, an antibacterial agent and / or an antifungal agent can also be added.
By culturing cells or tissues in such an aqueous sol, the cells or tissues are labeled with the complex. The dose of the complex in this case is preferably about 75 to 300 μg per 1 × 10 ⁶ cells in terms of the metal mass of the magnetic metal oxide. As the culture conditions, appropriate conditions can be selected depending on the type of cells or tissues. In the case of islet cells, the cells are usually cultured at 30 to 39 ° C. and 2 to 5% CO ² for 0.5 to 24 hours.

In addition, when transplanting cells or tissues labeled with the complex of the present invention and conducting MRI diagnosis, it is desirable to transplant 1 × 10 ⁶ or more labeled cells.

<1> Synthesis of Complex of Cationic Polysaccharide Derivative and Magnetic Metal Oxide (A) Preparation of Cationic Polysaccharide Derivative 100 g of various polysaccharides are dissolved in 100 mL of water, and sodium hydroxide and a cationizing agent (DEAE) are dissolved therein. -Cl: diethylaminoethyl chloride or SY-GTA80 (manufactured by Sakamoto Yakuhin Kogyo Co., Ltd .: glycidyltrimethylammonium chloride) is added at about 30 ° C. or lower, and then stirred at about 60 ° C. for 2 to 3 hours. Add 100 mL of water, cool, and adjust the pH to 8 by adding hydrochloric acid. Depending on the polysaccharide and cation group substitution rate used, 1.5 to 2.5 times the methanol and acetone of the reaction solution are added with stirring to precipitate the desired product. The operation of re-dissolving the precipitate in 500 mL of water and adding methanol and acetone to precipitate the target product was further repeated three times, and the resulting precipitate was dissolved in 500 mL of water and adjusted to pH 8 using sodium hydroxide. Filtration through a glass filter, concentration under reduced pressure, and lyophilization yield a cationic polysaccharide (hydrochloride).
Table 1 shows the raw materials and reagents used, and the yield and degree of substitution of the cationic polysaccharide derivative.

(B) Synthesis of complex [Examples 6, 9, and 10, Comparative Examples 1 and 2]
Polysaccharide No. 1 prepared in (A). The cationic polysaccharides 11.3 to 22.6 g of 1, 3, 4, 5, and 7 were dissolved in 40 mL of water and purged with nitrogen at room temperature with stirring. To this was added a mixed iron salt solution in which 0.9 g of ferrous chloride tetrahydrate was dissolved in 8.8 mL of 1M ferric chloride solution, and 1.5 N sodium hydroxide solution was further added to about pH 11. Next, hydrochloric acid was added to adjust the pH, followed by centrifugation. In Comparative Example 2, since a large amount of precipitate was generated by centrifugation, the subsequent operation was not performed.
The obtained solution was purified by ultrafiltration purification using distilled water or a borate buffer of pH 9 as a solvent, and preferential precipitation of the complex by addition of acetone to sufficiently remove free cationic polysaccharides and salts. The resulting solution was filtered through a membrane filter (pore size 0.20 μm) and filled into an ampoule.

[Examples 1-5, 7, 8]
Polysaccharide No. 1 prepared in (A). 1 to 6 and 8 cationic polysaccharides 11.3 to 22.6 g were dissolved in 40 mL of water and purged with nitrogen at room temperature with stirring. To this was added a mixed iron salt solution in which 0.9 g of ferrous chloride tetrahydrate was dissolved in 8.8 mL of 1M ferric chloride solution, and 1.5 N sodium hydroxide solution was further added to about pH 11. Next, hydrochloric acid was added to adjust the pH, and the mixture was heated to reflux at 100 to 103 ° C. for 1.5 hours, cooled and centrifuged. The obtained solution was purified by ultrafiltration purification using distilled water or a borate buffer of pH 9 as a solvent, and preferential precipitation of the complex by addition of acetone to sufficiently remove free cationic polysaccharides and salts. The resulting solution was filtered through a membrane filter (pore size 0.20 μm) and filled into an ampoule.

[Comparative Example 3]
The above polysaccharide No. 86 g of polysaccharide derivative 9 was dissolved in 240 mL of water and purged with nitrogen while heating to 80 ° C. with stirring. To this was added a mixed iron salt solution in which 18 g of ferrous chloride tetrahydrate was dissolved in 184 mL of a 1M ferric chloride solution, and 3N sodium hydroxide solution was further added to about pH11. Next, after adding hydrochloric acid to adjust to pH 7, the mixture is heated to reflux at 100 to 103 ° C. for 1.5 hours, cooled and then centrifuged. The resulting solution was purified by ultrafiltration. The solution was filtered through a membrane filter (pore size 0.20 μm) and filled into an ampoule.
Table 2 summarizes the polysaccharide derivatives used, the presence or absence of heating reflux, and the mass ratio of the polysaccharide derivative and magnetic iron oxide.
The ratio between the cationic polysaccharide derivative and the magnetic metal oxide was measured by the method described above.

<2> Complex stability test Next, D-MEM (Gibco BRL, Dulbecco's Modified Eagle Medium, Model No. 11995-065 (hereinafter referred to as “serum-containing D”) containing 20% fetal bovine serum (FBS). The stability of the complex was tested using an antimicrobial / antifungal agent (Penicillin-Streptomycin, liquid, product number 15140-122, manufactured by Gibco BRL). ) 1% was added.
The procedure is shown below.

(1) Measurement of degree of aggregation Each complex was diluted with a borate buffer of pH 9.0 so that the concentration was 0.05 mg / mL in terms of iron mass to obtain an aqueous sol. The average primary particle diameter was measured by a scattering method (for example, see Polymer J., 13, 1037-1043 (1981)). The measurement conditions are as follows.
(Measurement condition)
Device name: Autosizer 4700 (Malvern), temperature: 25 ° C., measurement angle: 90 ° C., wavelength: 514 nm
Subsequently, each complex was converted to iron mass, diluted with serum-containing D-MEM to a concentration of 0.10 mg / mL, allowed to stand in a 37 ° C. incubator for 24 hours, and then with distilled water. Diluted to 1/2 to obtain an aqueous sol, the average size of the aggregate was measured by the same method and conditions as described above.
The degree of aggregation of each composite was calculated by dividing the measured value of the average size of the aggregate by the measured value of the average primary particle diameter. The results are shown in Table 3.

(2) For the T ₂ relaxation ability aqueous sol of serum-containing D-MEM was prepared in the measurement (1) of a pulse NMR of 20 MHz (magnetic field is about 0.5 tesla), measured the T ₂ relaxation time, T ₂ Mitigation ability was calculated. The T ₂ relaxation time was measured 2 hours, 5 hours, and 24 hours after preparation of the aqueous sol. The method for calculating the T ₂ relaxation ability is as described above.
Separately, a water sol and an aqueous sol were prepared using distilled water and serum-free D-MEM as a solvent, and the T ₂ relaxation ability was similarly calculated. Note that the water sol, was also measured its the T ₂ relaxation ability immediately after production (0 hours).
The results are shown in Table 3.

The complexes of Examples 1 to 8 and Comparative Example 3 had a low degree of aggregation in serum-containing D-MEM. In particular, the complexes of Examples 1, 2, 4, and 7 had very low aggregation in serum D-MEM. On the other hand, the complex of Comparative Example 1 had a high degree of aggregation in serum-containing D-MEM.
In addition, the composites of Examples 1 to 8 and Comparative Example 3 were stable in T ₂ relaxation ability even after a certain period of time in serum-containing D-MEM. On the other hand, the complex of Comparative Example 1 could not measure T ₂ relaxation ability in serum-containing D-MEM.
From the above, it was found that a complex having an aggregation degree of 4.0 times or less in serum-containing D-MEM has a stable T ₂ relaxation ability even after a certain period of time.
In addition, it was found that it is effective to alcoholate the terminal aldehyde of the raw material polysaccharide in order to obtain a complex having an aggregation degree of 4.0 times or less.
Further, from the comparison of the T ₂ relaxation ability of the composites of Examples 5 and 6, it was found that the T ₂ relaxation ability was improved by heating and refluxing.

<3> Cell Labeling Test of Complex The complexes of Examples 1 to 4 and Comparative Examples 1 and 3 were combined with cancer cells derived from mouse islets (cell line: MIN6) (Jun-ichi Miyazaki et al., Endocrinology, Vol. 127, No. 1, p. 126-132. In the present specification, the amount of cell labeling was examined by coexisting with “MIN6” in some cases. Cell culture and labeling were performed as follows.
MIN6 was cultured in a 75 cm ² culture flask containing the serum-containing D-MEM under conditions of 37 ° C. and 5% CO ₂ , and the medium was changed every 3 days. The cells were washed with phosphate buffered saline (pH 7.4) (Ambion) (hereinafter, PBS), PBS in which trypsin (Gibco BRL) was dissolved was added, the cells were suspended, and then the same amount of serum. Containing D-MEM was added to inactivate trypsin. The supernatant was removed by centrifugation at 1200 rpm (240 × g) for 3 minutes, and the cells were collected. After washing the cells twice with PBS, the cells were prepared with serum-containing D-MEM to 2 × 10 ⁶ cells / 3 ml and seeded in a 60 mm ² culture dish.
Each complex was added to the culture dish so that the iron mass was 300 μg per 1 × 10 ⁶ cells, and cultured at 37 ° C. under 5% CO ₂ for 1 hour. The obtained culture broth was centrifuged at 1200 rpm (240 × g) for 3 minutes to remove serum-containing D-MEM. Cells were prepared to 1 × 10 ⁶ and washed twice with PBS. The obtained pellet-like cells were suspended in 1 mL of distilled water, homogenized, transferred to a measuring tube, and stored at 4 ° C. Thereafter, the T ₂ relaxation time of the cell suspension was measured by the method described above.
Further, based on the measured T ₂ relaxation time, the concentration of the complex in the cell suspension after each incubation time (iron mole) based on the T ₂ relaxation ability of the water sol (0 hour) measured in <2> (2). (Concentration conversion) was calculated.
Subsequently, the total concentration of the complex is calculated by multiplying the concentration of the complex by 1 mL of the liquid volume, and this is converted into mass, and the labeled amount per 10 ⁶ cells (μg-Fe / 1 × 10 ⁶ cells). ).
The results are shown in Table 4.

In the complexes of Examples 1, 2, 4, and Comparative Example 1, the amount of cell labeling after 1 hour of culture was as large as 0.6 μg-Fe / 1 × 10 ⁶ cells or more. The amount of cell labeling of the complex of Example 4 was small because the degree of substitution of the cationic group of the polysaccharide was small and the positive charge was relatively small. The cell labeling amount of the complex of Comparative Example 3 was small because the polysaccharide was not substituted with a cationic group and had a negative charge.
From this, when using the composite_body | complex of this invention as a labeling agent of a cell, it turned out that it is preferable that the substitution degree of a cation group exists in the range of 0.2-0.6 mol / AGU.
The reason that the cell labeling amount in Comparative Example 1 was extremely large is presumed to be that the aggregates of the complex adhered to the cell surface. Assuming that the complex is used as a labeling agent for MRI diagnosis of transplanted cells, if the aggregate is attached to the cell surface, there is a high possibility that the complex will be detached in vivo after cell transplantation, There is a high possibility that an accurate diagnosis cannot be made.

<4> Examination of Cell Labeling Conditions—Medium Using the complex of Example 1, the effect of medium conditions on the amount of MIN6 cell labeling was examined.
In the same manner as above, MIN6 was prepared using serum-containing D-MEM and seeded in a 60 mm ² culture dish. On the other hand, MIN6 was similarly prepared using D-MEM not containing serum (serum-free D-MEM), and seeded in a culture dish of 60 mm ² . To each culture dish, the complex of Example 1 (300 μg-Fe / 1 × 10 ⁶ cells) was added, and cultured for 30 minutes at 37 ° C. and 5% CO ₂ . Serum-containing D-MEM and serum-free D-MEM were removed by centrifugation at 1200 rpm (240 × g) for 3 minutes. Cells were prepared to 1 × 10 ⁶ and washed twice with PBS. The obtained pellet-like cells were suspended in 1 mL of distilled water, homogenized, transferred to a measuring tube, and stored at 4 ° C. Thereafter, the amount of cell labeling was calculated by the above method.
The results are shown in Table 5.

  From this, it was found that the amount of labeling was improved by adding serum to the culture medium used for cell labeling.

<5> Examination of Cell Labeling Conditions—Time Using the complex of Example 1, the effect of labeling time on the amount of cell labeling of MIN6 was examined. The labeling conditions other than the time were the same as in <3> above. After adding the complex to the culture dish, the cells were cultured for 0.5 hour, 1 hour, and 2 hours, respectively. Serum-containing D-MEM was removed by centrifugation at 1200 rpm (240 × g) for 3 minutes. Cells were prepared to 1 × 10 ⁶ and washed twice with PBS. The obtained pellet-like cells were suspended in 1 mL of distilled water, homogenized, transferred to a measuring tube, and stored at 4 ° C. Thereafter, the amount of cell labeling was calculated by the method described above.
Table 6 shows the results.

  From this, it was found that the labeling time of the cells is preferably about 0.5 hours to 2 hours, and most preferably about 1 hour.

<6> Examination of Cell Labeling Conditions—Dose Using the complex of Example 1, the effect of the complex dose on the MIN6 cell labeling amount was examined. The culture time was 0.5 hours, and the complexes were 15 μg-Fe / 1 × 10 ⁶ cells, 75 μg-Fe / 1 × 10 ⁶ cells, 150 μg-Fe / 1 × 10 ⁶ cells, 300 μg-Fe, respectively. / 1 × 10 ⁶ cells were tested. Other labeling conditions were the same as described above. Serum-containing D-MEM was removed by centrifugation at 1200 rpm (240 × g) for 3 minutes. The cells were adjusted to 1 × 10 ⁶ and washed twice with PBS. The obtained pellet-like cells were suspended in 1 mL of distilled water, homogenized, transferred to a measuring tube, and stored at 4 ° C. Thereafter, the amount of cell labeling was calculated by the method described above.
Table 7 shows the results.

From this, it was found that the dose of the complex is preferably about 75 to 300 μg in terms of the mass of the magnetic metal oxide per 1 × 10 ⁶ cells.

<7> Cytotoxicity test MIN6 was labeled using the conjugates of Examples 1 and 3 and Comparative Example 1, and cytotoxicity was measured by Trypan blue exclusion procedure. The labeling conditions were the same as in <3> above. A method for measuring cytotoxicity is shown below. Trypan blue solution (manufactured by Wako Pure Chemical Industries, Ltd.) was added to a sample in which pellet-like cells were suspended in 1 mL of PBS, and the cell viability was measured. As a control experiment, cell viability was similarly measured for cells not labeled with the complex.
Table 8 shows the results.

As a result, no cytotoxicity was observed in any complex.
In addition, a suspension of MIN6 for control, a suspension of MIN6 labeled with the complex of Example 1, and an aqueous sol of the complex of Example 1 (300 μg-Fe / mL, solvent: serum-containing D-MEM medium) ) Is centrifuged at 1200 rpm, 3 min, 4 ° C., and a photograph is shown in FIG. When the suspension of MIN6 labeled with the complex of Example 1 was centrifuged, cells colored reddish brown (appearing black in the figure) were precipitated. This revealed that MIN6 was labeled with the complex of Example 1. On the other hand, in the control containing no complex, MIN6 precipitated and looked whitish. In addition, no precipitation was observed in the aqueous sol of the composite.
As a result, it was revealed that islet cells are labeled by the complex of the present invention.

<8> Evaluation of MRI of labeled cells Precipitation of MIN6 labeled with the complex of Example 1 after the centrifugation and control MIN6 were observed by nuclear magnetic resonance imaging (MRI). The result is shown in FIG.
MIN6 labeled with the complex of the present invention was imaged by MRI. As a result, it was revealed that MIN6 labeled with the complex of the present invention can be observed by MRI.

<9> Implantation of MIN6 into mice and MRI evaluation After MIN6 was labeled with the complex of Example 1, the labeled MIN6 was placed under the left renal capsule of mice (C57BL / 6Cr, 7 weeks old, 25 g, male). The cells were transplanted and evaluated for the behavior of the cells transplanted in the body. MIN6 was labeled under the conditions described in <3> above. The labeled MIN6 was adjusted with physiological saline so that the cell concentration was 5 × 10 ⁶ cells / 10 μL, and MIN6 was transplanted under the kidney capsule of the mouse. 24 hours after transplantation, the behavior of the transplanted cells in the mouse body was evaluated by MRI. As a control experiment, a similar experiment was performed on mice transplanted with MIN6 not labeled with a complex. The result is shown in FIG.

  As a result, it was revealed by MRI that magnetically labeled islet cells can be observed at the transplantation site under the kidney capsule in the mouse body.

<10> Transplantation of fresh rat islet cells into mice and MRI evaluation Isolation of islet cells was performed from rats by the method described in Joo Ho Tai et al., Diabetes, Vol. 55, November 2006.
Rat islet cells separated by the complex of Example 1 were labeled, and then transplanted under the renal capsule of athymic mice (nuBalb / c, 7 weeks old, 25 g, male) to evaluate the behavior of the transplanted cells. It was. The method of transplantation and MRI was referred to Joo Ho Tai et al., Diabetes, Vol. 55, November 2006. Rat islet cells were labeled according to the conditions described in <3> above. The labeled cells were adjusted with physiological saline so that the cell concentration was 5 × 10 ⁶ cells / 10 μL, and transplanted under the kidney capsule of mice. 24 hours after transplantation, the behavior of the transplanted cells in the mouse body was evaluated by MRI. As a control experiment, mice transplanted with islet cells that were not labeled with the complex were similarly evaluated.
The results are shown in FIG.

As shown in FIG. 4, magnetically labeled islet cells could be observed at the transplantation site under the renal capsule in the mouse body.
From the above, it was revealed that magnetically labeled islet cells can be observed by MRI at the transplantation site under the renal capsule in the mouse body.

In recent years, diabetes has become a national disease in Japan, but there are about 140,000 people with severe type 1 diabetes, whose hypoglycemic attacks of acute complications are life-threatening, and nephropathy of chronic complications is renal failure. More than half die in 5 years. Currently, there are only pancreatic transplantation and pancreatic islet transplantation after isolating Ra islet from the pancreas. Pancreas transplants have a one-year graft survival rate of 80% and can completely cure type 1 diabetes, but require relatively large surgery and have approximately 10% surgical complications. On the other hand, islet transplantation involves only injecting isolated islet cells via the portal vein and does not require surgical operation, and can be performed even in severely diabetic patients. In 2000, 100% insulin withdrawal was reported using a new immunosuppressive drug at the University of Alberta in Edmonton, Canada (Edmonton Protocol). After this report, the reproducibility of the Edmonton protocol has been confirmed in various facilities in Europe and the United States.
A cationic polysaccharide magnetic particle complex having high uptake efficiency into cells and low cytotoxicity is considered to be effective for imaging transplanted cells in islet transplantation.

It is a photograph showing a cell mass of MIN6 labeled with the complex of the present invention (left), a cell mass of MIN6 not labeled with the complex (center), and an aqueous sol of the complex (right). The black portion of the center figure is a cell mass colored reddish brown. MRI images of labeled MIN6 cell mass in a complex of the present invention (T ₂ condition), and a composite in MIN6 MRI images that are not labeled (T ₂ conditions). Solvents and cells are contrasted white and when relaxed with the complex, they are contrasted black. It can be confirmed that the cell mass labeled with the complex is contrasted black. After transplanting MIN6 labeled with the complex of the present invention under the left renal capsule of a mouse, MRI images (T2 conditions) for evaluating the behavior of transplanted cells in the body, and MIN6 not labeled with the complex were similarly used. It is an MRI image (T2 condition) when transplanted. The cells are contrasted white and contrasted black when relaxed by the complex. It can be confirmed that the cells labeled with the particles (arrow portions) are contrasted black on the ventral side under the left renal capsule. After the rat islet cells labeled with complexes of the present invention was transplanted under the left renal coating athymic mice, MRI image (T ₂ condition) of the evaluation of the behavior of transplanted cells in the body, and are labeled with complex when implanted in the same manner without rat pancreatic islet cells is an MRI image (T ₂ condition) of. The cells are contrasted white and contrasted black when relaxed by the complex. It can be confirmed that the cells labeled with the particles (arrow portions) are contrasted black on the dorsal side under the left renal capsule.

Claims (17)

A complex of a cationic polysaccharide derivative and a magnetic metal oxide having the following properties:
The degree of aggregation is 4.0 times or less when left in a D-MEM containing 20% fetal bovine serum under conditions of 37 ° C. and 5% CO ₂ for 24 hours.   The complex according to claim 1, wherein the terminal aldehyde of the cationic polysaccharide derivative is carboxylated or alcoholated.   The composite according to claim 1 or 2, which is obtained by coating a magnetic metal oxide with a cationic polysaccharide derivative and then heating at 50 to 120 ° C for 5 minutes to 6 hours.   The composite according to any one of claims 1 to 3, wherein the magnetic metal oxide is ferrite.   The complex according to any one of claims 1 to 4, wherein the cationic polysaccharide derivative is an aminoalkyl etherified polysaccharide.   The complex according to any one of claims 1 to 5, wherein the polysaccharide is at least one selected from dextran, dextrin, cellulose, agarose, starch and pullulan.   The complex according to any one of claims 1 to 6, wherein the substitution degree of the cationic polysaccharide derivative is within a range of 0.2 to 0.6 mol / AGU. The composite according to any one of claims 1 to 7, which has the following properties:
When allowed to stand at 37 ° C. and 5% CO ₂ for 1 hour in D-MEM containing 20% fetal calf serum containing islet cells and a complex sufficient for the cells, the complex The labeled amount per 1 × 10 ⁶ cells is 0.6 μg or more in terms of the metal mass of the magnetic metal oxide.   The composite according to any one of claims 1 to 8, comprising the cationic polysaccharide derivative within a range of 0.05 to 20 parts by mass per 1 part by mass of the metal in the magnetic metal oxide.   The composite according to any one of claims 1 to 9, wherein the average primary particle diameter measured by a dynamic light scattering method is in the range of 5 to 100 nm. The composite according to any one of claims 1 to 10, wherein the T ₂ relaxation ability in the form of a water sol is in the range of 90 to 1000 (mM · sec) ^-1 .   The complex according to any one of claims 1 to 11, which is a labeling agent for cells or tissues.   The complex according to claim 12, wherein the cell or tissue is an islet cell or an islet.   The complex according to claim 12 or 13, which is an MRI contrast agent.   The method for producing a composite according to any one of claims 3 to 14, comprising heating the magnetic metal oxide with a cationic polysaccharide derivative and then heating at 50 to 120 ° C for 5 minutes to 6 hours.   The method for producing a cationic polysaccharide derivative according to claim 15, wherein a terminal aldehyde of the polysaccharide is carboxylated or alcoholated.   A method for labeling a cell, comprising culturing the cell in the presence of the complex according to any one of claims 1 to 14.