JP2007220867A - Water-dispersible magnetic nanoparticle - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide water-dispersible magnetic nanoparticles which have physiologically active material on their surfaces, and to provide a method of manufacturing the above magnetic nanoparticles which is simple and high in general-purpose properties. <P>SOLUTION: The magnetic nanoparticles are dispersed in water with 1 to 50 nm average grain diameter, and acid polysaccharide is fixed on their surfaces. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to water-dispersible magnetic nanoparticles for use in fields such as life science or medical diagnosis. More specifically, the present invention relates to water-dispersible magnetic nanoparticles having an acidic polysaccharide immobilized on the surface.

Affinity beads for isolating and immobilizing receptors for drugs and chemical substances have been developed and applied, but there is a possibility that applications can be further expanded by imparting magnetic responsiveness to these beads. For example, Non-Patent Document 1 describes synthesizing ferrite magnetic nanoparticles with functionality as HTS beads, and uses such magnetic nanoparticles as an MRI contrast agent, DDS, sensor, and the like. The application of is described. Non-Patent Document 2 describes a method for synthesizing ferrite (solid solution of Fe ₃ O ₄ and δ-Fe ₂ O ₃ ) nanoparticles in a reaction solution at a low temperature of 4 ° C. and near neutrality. Non-Patent Document 2 shows that by containing proteins and various physiologically active substances in the reaction solution, they can be firmly fixed to the surface of the ferrite particles by chemical bonding during the synthesis of the ferrite particles. Yes. By applying this technology, ferrite nanoparticles are firmly coated with polymer molecules, and bioactive substances such as antibodies and drugs are strongly immobilized without losing their activity, aiming at high-speed bioscreening and other new applications. ing.

  In biotechnology, nanoparticles are expected to be used in a wide range of fields such as separation of cells, DNA and proteins, various assays, diagnosis and drug delivery systems (DDS). For example, Non-Patent Document 3 describes the production of thermoresponsive magnetic nanoparticles by combining thermoresponsive polymers as magnetic nanoparticles, and is applied to bioseparation, from genetic engineering to genome / proteome analysis. Applications and cell separation / assay applications are described. Non-Patent Document 3 is expected to greatly improve sensitivity and shorten measurement time simply by replacing the magnetic fine particles and latex carrier used in conventional analysis methods with Therma-Max, a temperature-responsive magnetic nanoparticle. It is described that it was made.

  In Non-Patent Document 4, when a ferrite nanoparticle is synthesized at a low temperature of 4 ° C., avidin is firmly fixed to the surface of the ferrite particle by chemical bonding by allowing avidin-FITC to coexist in the reaction solution. It is shown that the pattern of the magnetic material and the fluorescence pattern coincide with each other by applying X-ray fluorescence on a flexible disk.

  Hydrophilic magnetic particles having a size of μm to several hundreds of nanometers are widely used as means for separating and purifying biological materials (proteins, nucleic acids, etc.), and the captured biomolecules can be easily magnetically separated. These magnetic particles exist in a suspended state in water, and the interaction with the target molecule is a heterogeneous reaction. If the size of the magnetic particles is reduced to several tens of nm or less to form a hydrophilic surface, a uniform dispersion can be obtained, and acceleration of interaction with biomolecules can be expected. Moreover, the possibility of application to medical diagnosis, treatment, etc. is expanding by controlling and detecting nano-sized particles by magnetism. Therefore, studies on the synthesis of magnetic nanoparticles and the water dispersion method have been actively conducted.

  As a means of uniformly dispersing water in the synthesized magnetic nanoparticles without forming aggregates, hydrophilic compounds are linked to the surface by covalent bonds, the surface is hydrophilized after embedding a polymer matrix such as polystyrene or silica, and coated with phospholipid. Alternatively, liposome inclusion, introduction of hydrophilic groups by silane coupling treatment, dextran coating or synthetic hydrophilic polymer coating such as polyacrylic acid are known. These cover the surface of magnetic nanoparticles and introduce a molecule such as an antibody that can interact with the target physiologically active substance by introducing a functional group on the surface. Therefore, a multi-step process is required to obtain water-dispersible magnetic nanoparticles having functionality. In addition, biocompatibility is not sufficient when the prepared magnetic particle surface is brought into direct contact with living tissue or cells. Solder et al. Have reported surface modification methods via connector molecules, but require precise particle formation with temperature control. Furthermore, a method of dispersing surface-treated magnetic particles in a water-soluble polymer solution such as carboxymethylcellulose or polyisopropylacrylamide is also known, and some are used for MRI contrast agents and the like.

  However, nano-particles that have a biocompatible surface and that can be linked to biologically active substances and that can be expected to interact with biologically derived molecules and that form a highly functional superparamagnet that forms a transparent aqueous dispersion. So far it has not been reported.

Construction of functional magnetic nanobeads and application to biotechnology, BIO INDUSTRY, Vol.21, No.8, 21-30, 2004, Nobuyuki Goemon, Hirosuke Nishio, Hiroshi Handa New production technology for magnetic nano-beads for medical use, BIO INDUSTRY, Vol.21, No.8, 7-13, 2004, Masaki Abe, Hiroshi Handa Development of magnetic nanoparticle material and its application to biotechnology, Journal of Japanese Society of Agricultural Chemistry, Vol.77, N0.9, 861-864, 2003, Akihiko Kondo, Noriyuki Onishi Direct immobilization of fluorescent dyes onto ferrite nanoparticles during their synthesis from aqueous solution, J. Appl. Phys., 93 (10), 7569-7570 (2003), M. Abe, H. Handa, et al.

  An object of the present invention is to solve the above-described problems of the prior art. That is, an object of the present invention is to provide a water monodispersed magnetic nanoparticle having a physiologically active substance on the surface and a versatile and simple production method of the above magnetic nanoparticle.

  As a result of diligent investigations to solve the above problems, the present inventors have mixed a magnetic nanoparticle dispersion and an acidic polysaccharide solution, and carried out ultrasonic irradiation, whereby water dispersibility in which the surface is substituted with acidic polysaccharide. It has been found that magnetic nanoparticles can be produced. The present invention has been completed based on this finding.

  That is, according to the present invention, there is provided a magnetic nanoparticle having an average particle size of 1 to 50 nm dispersed in water, wherein an acidic polysaccharide is immobilized on the surface. The

Preferably, the magnetic nanoparticles dispersed in water have the general formula R ¹ — (OCH ₂ CH ₂ ) _n —OLX (wherein R ¹ represents an alkyl group having 1 to 24 carbon atoms, n represents an integer of 1 to 20, L represents a single bond or an alkylene group having 1 to 10 carbon atoms, and X represents a carboxylic acid group, a phosphoric acid group, a sulfonic acid group, or a boric acid group). Surface modified with a compound
Preferably, the magnetic nanoparticles having an average particle diameter of 1 to 50 nm are iron oxide or ferrite.

Preferably, the acidic polysaccharide to be immobilized is an acidic polysaccharide containing uronic acid.
Preferably, the acidic polysaccharide to be immobilized is selected from hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, chondroitin polysulfate, heparin, or heparan sulfate.

  According to another aspect of the present invention, magnetic nanoparticles having an average particle size of 1 to 50 nm dispersed in water are treated by irradiating with ultrasonic waves in the presence of acidic polysaccharide. A method for producing magnetic nanoparticles having a polysaccharide immobilized thereon is provided.

Preferably, the ultrasonic irradiation is performed in a buffer solution having a pH of 5.0 or more.
Preferably, the ultrasonic irradiation is performed in a phosphate buffer.
Preferably, the irradiation time of ultrasonic waves is 1 minute or more and 2 hours or less.
Preferably, an ultrasonic wave having a high frequency output of 0.1 to 200 W is applied.

  According to still another aspect of the present invention, there are provided magnetic nanoparticles produced by the method of the present invention described above.

  According to still another aspect of the present invention, there are provided a thermotherapy agent, an MRI contrast agent, a drug delivery agent, an analytical diagnostic probe, and a physiologically active substance separating agent comprising the above-described magnetic nanoparticles of the present invention. .

  According to still another aspect of the present invention, there is provided a method for magnetic separation and purification of a physiologically active substance, which comprises bringing the magnetic nanoparticles of the present invention into contact with the physiologically active substance.

  According to the present invention, it is possible to easily introduce a target physiologically active substance into magnetic nanoparticles with few steps while maintaining water dispersibility. The magnetic nanoparticles of the present invention have a biocompatible surface, can be linked to physiologically active substances, and can be expected to interact with biological molecules. The magnetic nanoparticles of the present invention have a good water dispersion and high performance superparamagnetism.

Hereinafter, embodiments of the present invention will be described in detail.
The magnetic nanoparticles of the present invention are magnetic nanoparticles having an average particle diameter of 1 to 50 nm dispersed in water, and characterized in that acidic polysaccharides are immobilized on the surface.

  The dispersion of magnetic nanoparticles used in the present invention is obtained, for example, by adding an aqueous solution of a surfactant (for example, polyoxyethylene (4,5) lauryl ether acetic acid) to an aggregate of magnetic nanoparticles and dispersing it. A dispersion of magnetic nanoparticles can be prepared. However, the method for preparing a dispersion of magnetic nanoparticles is not limited to this. For example, a hydrophilic polymer [polyethylene glycol, sodium polyphosphate, etc.] or a phospholipid (phosphatidylcholine, etc.) is synthesized or synthesized. Words may coexist.

  In the present invention, it is preferable to use magnetic nanoparticles having an independently dispersed average particle diameter of 1 to 50 nm and having an anionic functional group on the surface. “Independently dispersed” means a state in which particles are dispersed alone in a solution without forming an aggregate. The average particle size of the magnetic nanoparticles is 1 to 50 nm, more preferably 1 to 40 nm or less, and particularly preferably 1 to 30 nm or less.

As magnetic nanoparticles, any particles can be used as long as they can be dispersed or suspended in an aqueous medium and can be separated from the dispersion or suspension by application of a magnetic field. Examples of the magnetic nanoparticles used in the present invention include iron, cobalt or nickel salts, oxides, borides or sulfides; rare earth elements having high magnetic susceptibility (eg, hematite or ferrite). As a specific example of the magnetic nanoparticles, for example, a ferromagnetic ordered alloy such as magnetite (Fe ₃ O ₄ ), FePd, FePt, CoPt, or the like can be used. Preferred magnetic nanoparticles in the present invention are those selected from the group consisting of metal oxides, in particular iron oxide and ferrite (Fe, M) ₃ O ₄ . Here, iron oxide includes, among others, magnetite, maghemite, or a mixture thereof. In the above formula, M is a metal ion that can be used together with the iron ion to form a magnetic metal oxide, and is typically selected from transition metals, most preferably Zn ²⁺ , Co ²⁺ , Mn ²⁺ , Cu ²⁺ , Ni ²⁺ , Mg ^2+, etc., and the molar ratio of M / Fe is determined according to the stoichiometric composition of the selected ferrite. The metal salt is supplied in solid form or in solution, but is preferably a chloride salt, bromide salt, or sulfate salt. Among these, iron oxide and ferrite are preferable from the viewpoint of safety. Particularly preferred is magnetite (Fe ₃ O ₄ ).

  The magnetic nanoparticles used in the present invention have an anionic functional group on the surface. Examples of the anionic functional group include a carboxylic acid group, a phosphoric acid group, a sulfonic acid group, and a boric acid group, and a carboxyl group is particularly preferable.

Preferably, magnetic nanoparticles whose surface is modified with a compound represented by the general formula R ¹ — (OCH ₂ CH ₂ ) _n —OLX can be used. In the formula, R ¹ represents an alkyl group having 1 to 24 carbon atoms, n represents an integer of 1 to 20, and L represents a single bond or a linear or branched alkylene group having 1 to 10 carbon atoms. , X represents a carboxylic acid group, a phosphoric acid group, a sulfonic acid group or a boric acid group.

  The magnetic nanoparticles of the present invention may contain a drug (pharmaceutically active ingredient) or may not contain a drug (pharmaceutically active ingredient).

  Acidic polysaccharides are immobilized on the surface of the magnetic nanoparticles of the present invention. The acidic polysaccharide to be immobilized is preferably an acidic polysaccharide containing uronic acid, and more specifically hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate D, chondroitin sulfate E, dermatan sulfate, chondroitin polysulfate, heparin. Or heparan sulfate is particularly preferred.

  The present invention further includes treatment of magnetic nanoparticles having an average particle size of 1 to 50 nm dispersed in water by irradiating ultrasonic waves in the presence of the acidic polysaccharide, wherein the acidic polysaccharide is immobilized on the surface. The present invention relates to a method for producing magnetic nanoparticles. In the present invention, by directly replacing the dispersant covering the surface of the water monodispersed magnetic nanoparticles in water, the target acidic polysaccharide can be easily introduced into the magnetic nanoparticles while maintaining water dispersibility. it can.

  In the present invention, the ultrasonic irradiation can be performed by a conventional method known to those skilled in the art, for example, using a commercially available ultrasonic bath. The ultrasonic irradiation is preferably performed in a buffer solution having a pH of 5.0 or more, and can be performed, for example, in a phosphate buffer solution. The irradiation time of ultrasonic waves is not particularly limited as long as the acidic polysaccharide can be immobilized on the surface of the magnetic nanoparticles, and can be appropriately set, and is generally from 1 minute to 2 hours. Also. As the ultrasonic wave, it is preferable to irradiate an ultrasonic wave having a high frequency output of 0.1 to 200 W.

  Since the magnetic nanoparticles of the present invention have magnetism, they can be guided to a predetermined site by magnetic force. That is, the magnetic nanoparticles of the present invention can be administered into the body and induced to the diseased site by magnetic force, and the magnetic nanoparticles induced to the diseased site as described above can be confirmed by MRI imaging. . That is, the magnetic nanoparticles of the present invention are useful as a contrast agent for MRI.

  The magnetic nanoparticles of the present invention may further contain a drug (pharmaceutically active ingredient) as desired. Such magnetic nanoparticles of the present invention can be induced to a diseased site according to the above method, and then heated by applying a high frequency to release the pharmaceutically active ingredient encapsulated in the nanoparticles. That is, the magnetic nanoparticles of the present invention are useful as a thermotherapy agent or a drug delivery agent.

  Furthermore, the magnetic nanoparticles of the present invention can also be used as an analytical diagnostic probe. Specifically, heparin or immobilized heparan sulfate-immobilized magnetic nanoparticles can be used for analytical diagnosis of various heparin-binding proteins (such as vitronectin and midkine). In addition, chondroitin sulfate-immobilized magnetic nanoparticles can be used for analysis and diagnosis of midkine, chemokine, L-selectin and the like.

  The kind of the pharmaceutically active ingredient that can be optionally contained in the magnetic nanoparticles of the present invention is not particularly limited, but is preferably an anticancer agent, an antiallergic agent, an antioxidant, an antithrombotic agent, an antiinflammatory agent, or the like. It is. Specific examples of anticancer agents that can be used in the present invention include fluorinated pyrimidine antimetabolites (5-fluorouracil (5FU), tegafur, doxyfluridine, capecitabine, etc.); antibiotics (mitomycin (MMC), adriacin (DXR), etc. ); Purine antimetabolite (antolefolate antimetabolite such as methotrexate, mercaptopurine etc.); active metabolite of vitamin A (antimetabolite such as hydroxycarbamide, tretinoin and tamibarotene); molecular targeting drug (herceptin and imatinib mesylate) Platinum preparations (Briplatin, Randa (CDDP), Paraplatin (CBDC), Elprat (Oxa), Akpra, etc.); Plant alkaloid drugs (Topotecin, Campto (CPT), Taxol (PTX), Taxotere (DTX), Etoposide Etc.); alkylating agent (busulfan) Anti-androgenic drugs (such as bicalutamide and flutamide); female hormone drugs (such as phosfestol, chlormadinone acetate, and estramustine phosphate); LH-RH drugs (such as Leuplin and Zoladex) Antiestrogens (tamoxifen citrate, toremifene citrate, etc.); aromatase inhibitors (fadrozol hydrochloride, anastrozole, exemestane, etc.); luteinizing hormone drugs (such as medroxyprogesterone acetate); BCG, etc. However, the present invention is not limited to these.

  In addition, a substance having selective affinity for cancer cells may be added to the magnetic nanoparticles of the present invention. Particularly preferably, an antibody or folic acid can be added. As an antibody having selective affinity for cancer cells, for example, an antibody that recognizes a cancer antigen can be used, and an antibody that recognizes a free antigen can be preferably used. Specific examples of cancer antigens include epidermal growth factor receptor (EGFR), estrogen receptor (ER), progesterone receptor (PgR) and the like.

  Antibodies having selective affinity for the above cancer cells can be easily obtained by those skilled in the art. For example, commercially available products may be used, or the above antigen or a partial peptide thereof may be used as an immunogen. It can be appropriately prepared and used by a known antibody preparation method. Further, the antibody to be used may be a monoclonal antibody or a polyclonal antibody.

  The above-described antibody is bound to the magnetic nanoparticle of the present invention by forming a peptide bond by an amidation reaction using an amino group or a carboxyl group that can be included in the magnetic nanoparticle of the present invention as a reactive group. Can do. The amidation reaction is performed by condensation of a carboxyl group or a derivative group thereof (ester, acid anhydride, acid halide, etc.) and an amino group. When an acid anhydride or acid halide is used, it is desirable that a base coexists. When an ester such as methyl ester or ethyl ester of carboxylic acid is used, it is desirable to perform heating or decompression in order to remove the generated alcohol. When directly amidating a carboxyl group, amidation reagents such as DCC, Morpho-CDI, and WSC, condensation additives such as HBT, N-hydroxyphthalimide, p-nitrophenyl trifluoroacetate, 2,4,5- A substance that promotes an amidation reaction such as an active ester such as trichlorophenol may be allowed to coexist or may be reacted in advance. Further, during the amidation reaction, it is desirable to protect either the amino group or the carboxyl group of the affinity molecule to be bound by amidation with an appropriate protecting group according to a conventional method, and to deprotect after the reaction.

  Nanoparticles to which an antibody having selective affinity for cancer cells by an amidation reaction is washed and purified by a conventional method such as gel filtration, and then water and / or a hydrophilic solvent (preferably methanol, ethanol, isopropanol, 2-ethoxyethanol or the like).

  The administration method of the magnetic nanoparticles of the present invention is not particularly limited, but it is preferably administered by injection into blood vessels, body cavities or lymph, and intravenous injection is particularly preferred.

  The dosage of the magnetic nanoparticles of the present invention can be appropriately set according to the weight of the patient, the state of the disease, etc. Generally, about 10 μg to 100 mg / kg is administered per administration. Preferably, about 20 μg to 50 mg / kg can be administered.

In addition, the magnetic nanoparticles of the present invention can be used to separate a physiologically active substance in a sample by contacting with the sample. That is, the magnetic nanoparticles of the present invention can be used as a separating agent for physiologically active substances, and depending on the biological sample, the magnetic nanoparticles can be brought into contact with the biological sample in the presence of an aggregation promoter. Here, the aggregation promoter is a substance that causes aggregation, and an appropriate substance can be used alone or in combination depending on the type of fraction to be aggregated.
The following examples further illustrate the present invention in detail but are not to be construed to limit the scope thereof.

Production Example 1: Preparation of magnetic nanoparticle dispersion 10.8 g of iron (III) chloride hexahydrate and 6.4 g of iron (II) chloride tetrahydrate were dissolved and mixed in 80 ml of 1N hydrochloric acid aqueous solution, respectively. While stirring this solution, 96 ml of aqueous ammonia (28 wt%) was added thereto at a rate of 2 ml / min. Thereafter, the mixture was heated at 80 ° C. for 30 minutes and then cooled to room temperature. The resulting aggregate was purified with water by decantation. Formation of magnetite (Fe ₃ O ₄ ) having a crystallite size of about 12 nm was confirmed by an X-ray diffraction method.

  To this agglomerate, 100 ml of an aqueous solution in which 2.3 g of polyoxyethylene (4,5) lauryl ether acetic acid (Nikko Chemicals) was dissolved (pH adjusted to 6.8 with NaOH) was added and dispersed to disperse the magnetic nanoparticles. The liquid was adjusted.

Example 1: Surface modification of magnetic nanoparticles with chondroitin sulfate C Magnetic nanoparticles (iron oxide content) dispersed in water with the surfactant (polyoxyethylene (4,5) lauryl ether acetic acid) produced in Production Example 1 Add 1.0 ml of 0.1M phosphate buffer (pH 7.6) containing 1% chondroitin sulfate C (Wako Pure Chemical Industries, Ltd.) to 1.0 ml of 8.4 g / L) dispersion, and use ultrasonic bath Sharp UT-105. Was irradiated with ultrasonic waves at 100W for 20 minutes. Collect the magnetic material aggregated with a magnet, remove the supernatant, add 1.0 ml of water, wash the aggregate with a vortex mixer, collect the aggregate again with a magnet, and discard the washing solution. Finally, 1.0 ml of water was added and ultrasonic irradiation was performed at 100 W for 5 minutes. As a result, the magnetic nanoparticles were uniformly redispersed and became a transparent dispersion. When the zeta potential of the redispersed magnetic nanoparticles was measured, it changed from −31 mV before treatment to −20 mV, and it was confirmed that the particle surface was replaced with chondroitin sulfate C.

Example 2: Surface modification of magnetic nanoparticles with hyaluronic acid Magnetic nanoparticles (iron oxide content 8) dispersed in water with the surfactant (polyoxyethylene (4,5) lauryl ether acetic acid) produced in Production Example 1 .4g / L) 1.0ml 0.1M phosphate buffer (pH7.6) containing 0.5% hyaluronic acid (manufactured by Wako Pure Chemical Industries, Ltd.) is added to 1.0ml of dispersion and 100W with ultrasonic bath Sharp UT-105. And then irradiated with ultrasonic waves for 20 minutes. Collect the magnetic material aggregated with a magnet, remove the supernatant, add 1.0 ml of water, wash the aggregate with a vortex mixer, collect the aggregate again with a magnet, and discard the washing solution. Finally, 1.0 ml of water was added and ultrasonic irradiation was performed at 100 W for 5 minutes. As a result, the magnetic nanoparticles were uniformly redispersed and became a transparent dispersion. When the zeta potential of the re-dispersed magnetic nanoparticles was measured, it changed from −31 mV before treatment to −16 mV, and it was confirmed that the particle surface was substituted with hyaluronic acid.

Example 3: Surface modification of magnetic nanoparticles with carboxymethylcellulose (CMC) Magnetic nanoparticles dispersed in water with the surfactant (polyoxyethylene (4,5) lauryl ether acetic acid) produced in Production Example 1 (oxidation) Add 1.0 ml of 0.1 M phosphate buffer (pH 7.6) containing CMC (manufactured by Wako Pure Chemical Industries, Ltd.) to 1.0 ml of a dispersion with an iron content of 8.4 g / L), and 100 W with an ultrasonic bath Sharp UT-105 Was irradiated with ultrasonic waves for 20 minutes. Collect the magnetic material aggregated with a magnet, remove the supernatant, add 1.0 ml of water, wash the aggregate with a vortex mixer, collect the aggregate again with a magnet, and discard the washing solution. Finally, 1.0 ml of water was added and ultrasonic irradiation was performed at 100 W for 5 minutes. As a result, the magnetic nanoparticles were uniformly redispersed and became a transparent dispersion. When the zeta potential of the re-dispersed magnetic nanoparticles was measured, it changed from −31 mV before treatment to −16 mV, and it was confirmed that the particle surface was replaced with CMC.

Claims (17)

A magnetic nanoparticle having an average particle diameter of 1 to 50 nm dispersed in water, wherein an acidic polysaccharide is immobilized on the surface. Magnetic nanoparticles dispersed in water have a general formula R ¹ — (OCH ₂ CH ₂ ) _n —OLX (wherein R ¹ represents an alkyl group having 1 to 24 carbon atoms, and n is 1 And an integer of 20 or less, L represents a single bond or an alkylene group having 1 to 10 carbon atoms, and X represents a carboxylic acid group, a phosphoric acid group, a sulfonic acid group or a boric acid group). The magnetic nanoparticles according to claim 1, wherein the magnetic nanoparticles are surface modified. The magnetic nanoparticle according to claim 1 or 2, wherein the magnetic nanoparticle having an average particle diameter of 1 to 50 nm is iron oxide or ferrite. The magnetic nanoparticle according to any one of claims 1 to 3, wherein the acidic polysaccharide to be immobilized is an acidic polysaccharide containing uronic acid. The magnetic nanoparticle according to any one of claims 1 to 4, wherein the acidic polysaccharide to be immobilized is selected from hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, chondroitin polysulfate, heparin, or heparan sulfate. Magnetic nanoparticles having an acidic polysaccharide immobilized on the surface, comprising treating magnetic nanoparticles having an average particle size of 1 to 50 nm dispersed in water by irradiating ultrasonic waves in the presence of the acidic polysaccharide Manufacturing method. The method according to claim 6, wherein the ultrasonic irradiation is performed in a buffer solution having a pH of 5.0 or more. The method according to claim 6 or 7, wherein the ultrasonic irradiation is performed in a phosphate buffer. The method according to any one of claims 6 to 8, wherein the ultrasonic irradiation time is 1 minute or more and 2 hours or less. The method according to claim 6, wherein an ultrasonic wave having a high-frequency output of 0.1 to 200 W is irradiated. Magnetic nanoparticles produced by the method according to claim 6. The thermotherapy agent containing the magnetic nanoparticle in any one of Claim 1 to 5 or 11. The MRI contrast agent containing the magnetic nanoparticle in any one of Claim 1 to 5 or 11. A drug delivery agent comprising the magnetic nanoparticles according to claim 1. An analytical diagnostic probe comprising the magnetic nanoparticles according to claim 1. A separation agent for a physiologically active substance, comprising the magnetic nanoparticles according to claim 1. A method for magnetic separation and purification of a physiologically active substance, comprising bringing the magnetic nanoparticles according to any one of claims 1 to 5 and 11 into contact with the physiologically active substance.