WO2005087367A1 - Magnetic composite particle and process for producing the same - Google Patents

Abstract

Magnetic composite particles of 0.01 to 4 μm average size comprising magnetic particles and, superimposed on surfaces thereof, chitosan layers. In particular, there are provided magnetic composite particles of core shell structure comprising magnetic particles as cores and chitosan layers as shells, which magnetic composite particles are capable of immobilizing physiologically active substances with high yield and after the immobilization, exhibit high collectability by magnetism. Further, there is provided a process for producing magnetic composite particle composed of magnetic particles and, superimposed on surfaces thereof, chitosan layers, which process comprises the steps of (a) dissolving chitosan in water by acidification; (b) dispersing magnetic particles in the chitosan solution and (c) slowly neutralizing the dispersion with a weak alkali base.

Description

 Specification

 Magnetic composite particles and method for producing the same

 Technical field

 [0001] This patent application claims the priority of Japanese Patent Application No. 2004-071889, which is hereby incorporated by reference in its entirety.

 The present invention relates to a magnetic composite particle for immobilizing a physiologically active substance, more specifically, a diagnostic drug carrier, a bacterial separation carrier, a nucleic acid separation and purification carrier, a protein purification carrier, an immobilized enzyme carrier, and an antibody immobilization carrier. The present invention relates to magnetic composite particles useful as carriers and the like.

 Background art

 [0002] Chitosan has conventionally been used for various purposes such as separation and purification because it has the property of adsorbing various substances. In addition, since chitosan has an amino group, its application to various fields has been attempted by immobilizing various functional groups and substances.

 In order to expand the applicability of chitosan, recently, an attempt has been made to composite chitosan and a magnetic substance into composite particles which can be easily separated and recovered by a magnetic field.

 [0003] For example, after dispersing magnetic fine particles in a chitosan solution in which chitosan is dissolved by making it acidic, a strong basic substance is dropped, or the above-mentioned chitosan solution is dropped into a strong basic substance to form composite particles. There is an attempt to obtain it (see Patent Document 1).

 In this example, the crosslinked chitosan granules have a structure enclosing a plurality of magnetic fine particles, and the chitosan that precipitates at the moment of contact with a strong basic substance entrains the surrounding magnetic fine particles and solidifies. It is thought to be. In addition, since one drop is basically one composite particle, a large composite particle cannot be obtained with respect to the magnetic fine particles. In order to improve the characteristics of the composite particles such as the adsorption efficiency, there are many requests to increase the surface area by increasing the size of the particles, but it is difficult to respond to this.

[0004] In order to improve the above points, an attempt has been made to prepare a dispersion liquid in which magnetic fine particles are dispersed in a chitosan solution and spray-dry the dispersion liquid (see Patent Document 2).

In this case, finer composite particles than in the previous example can be obtained, However, there is no change in including a plurality of magnetic fine particles. Also in the embodiment of this example, the size of the obtained chitosan composite particles is as large as 110 m with respect to the magnetic fine particle size of 0.01-0.5 m.

[0005] Also, attempts have been made to obtain composite particles by emulsifying a dispersion in which magnetic particles are dispersed in a chitosan solution and dispersing them in a chitosan solution! (See Patent Document 3). Similarly, there is no example using chitosan, but there is also an example in which gelatin is used to show that composite particles can be obtained by crosslinking after emulsification (see Patent Document 4). However, in these examples, since the magnetic particles are smaller than the composite particles, one composite particle includes a plurality of magnetic particles. In addition, since the composite particles are spherical, the ratio of chitosan other than the magnetic particles must be increased in consideration of the gap between the magnetic particles. It ’s not so good.

 [0006] As seen in the above examples, it is obvious that a structure in which a single composite particle includes a plurality of magnetic particles causes a thick portion and a thin portion of chitosan. When the composite particles are obtained, or when the chitosan in the thin portion is broken, the internal magnetic substance may be partially exposed. When the surface of the magnetic substance is exposed in this way, it is often undesirable, for example, that an unintended reaction occurs with the physiologically active substance.

 [0007] In some cases, as an attempt to obtain fine particles, coprecipitation or post-precipitation is used (see Patent Document 5). In the embodiment of this example, a post-precipitation technique is employed in which an acidic solution of chitosan is added to a superparamagnetic magnetite dispersion. In this case, even after the addition of the chitosan solution, the solution becomes strongly acidic, so that the coating is formed in a state where the chitosan is highly soluble, and as a result, the coating layer becomes a very thin layer of one molecule level. Is self-evident.

 Originally, this example reduced the chitosan ratio and did not assume that a thick coating would be formed. With such a thin coating, the internal magnetic substance may be exposed from the gaps between the adsorbed molecular chains, or the coating may be partially peeled off by mechanical force or the like to expose the internal magnetic substance.

[0008] As a similar example, a polymer layer having a reactive functional group is formed on the surface of magnetic particles by copolymerizing or seed-polymerizing a monomer having a reactive functional group. Attempts have been made to react an active group with chitosan (see Patent Document 6).

 Also in this case, since only the molecules that react with the surface cover the surface, it is obvious that the coating layer is a very thin layer of one molecule level, and the polymer may be exposed. Further, if a particle containing a plurality of magnetic particles is formed in the polymerization step of the monomer, a composite particle containing a plurality of magnetic particles is finally obtained, causing the same problem as described above.

 [0009] There are also scientific literatures examining the various methods described above. (1) A method in which magnetic fine particles are dispersed in a chitosan solution in which chitosan is dissolved by acidification, and then a strong basic substance is dropped, (2) A method in which a reactive functional group is introduced into the surface of a magnetic particle with a silane coupling agent, and the reactive functional group reacts with chitosan. (3) Mix raw materials of magnetic particles in a chitosan solution and add a strong basic substance Accordingly, a coprecipitation method for simultaneously producing magnetic particles and precipitating chitosan has been studied using 7-lOnm micromagnetic particles (see Non-Patent Document 1).

 [0010] As a result, as described above, (1) generates a large composite particle including a plurality of magnetic particles, and (2) indicates that the thin coating indicates that the ratio of the magnetic particle diameter to the chitosan is small. (Calculated from the amount of amino groups), and a plurality of magnetic particles are aggregated! Also, in (3), electron micrographs are shown in the literature, but no form was found in which chitosan covered the surface, indicating that the form was a mixture of magnetic fine particles and chitosan particles. To be observed. In fact, in the coprecipitation method, the formation of magnetic particles and the precipitation of chitosan proceed simultaneously, so it is thought that a layered structure is formed in which magnetic particles are formed and chitosan is deposited thereon. It is assumed that particles have been generated.

 Patent Document 1: Japanese Patent Publication No. 6-51114

 Patent Document 2: Japanese Patent Application Laid-Open No. 7-188062

 Patent Document 3: JP-T-59-500399

 Patent Document 4: Japanese Patent Publication No. 57-501411

 Patent Document 5: Tokiohei 10-506121

 Patent Document 6: JP-A-2002-17400

Non-patent Literature 1 Journal of Fermentation and Bioengineenng, Vol.86, No., 191-196 (1998)

 Disclosure of the invention

 Problems to be solved by the invention

[0012] In view of such a conventional technique, the present invention can fix a physiologically active substance in a high yield as a composite material of magnetic particles and chitosan, and can capture magnetically active material after the immobilization. High concentration! ヽ To provide composite materials! Puru.

 Means for solving the problem

 [0013] The present inventors have conducted intensive studies in order to achieve the above object, and as a result, used a weak alkali base as a reagent for chitosan precipitation, and gradually reduced the base to neutral. By adopting the specific method described above, composite magnetic particles having a chitosan layer on the surface of each of the magnetic particles can be generated. The present inventors have found that it is possible to fix the product in a high yield, and that the magnetism after the fixation is high, so that the present invention has been completed.

 That is, a desirable mode of the present invention also has the following constituent powers.

 1. Magnetic composite particles having a chitosan layer on the surface of magnetic particles and having an average particle size in the range of 0.01-.

 2. The magnetic composite particle according to the above item 1, having a core-shell structure in which a particle having magnetism is a core and a chitosan layer is a shell.

 3. The magnetic composite particle according to the first or second above, wherein the chitosan layer is crosslinked.

 4. The magnetic composite particle according to the above item 3, wherein a cross-linking agent having two or more functional groups selected from the group consisting of an aldehyde group, an epoxy group and an isocyanate group is used for cross-linking the chitosan layer.

 5. The magnetic composite particles according to any one of the above items 14 to 14, wherein the particles having magnetism are ferromagnetic oxide particles.

6. Ferromagnetic oxidized iron particles Magnetite particles, maghemite particles, magnetite-magtomite intermediate oxidized particles, gamma hematite alpha hematite intermediate oxidized iron particles, manganese zinc ferrite particles, rare earths The magnetic composite particles according to the fifth aspect, wherein the magnetic composite particles are at least one selected from the group consisting of iron garnet particles and bismuth-substituted rare earth iron garnet particles. 7. The magnetic composite particles according to the above item 5, wherein the particles having magnetism are particles obtained by coating ferromagnetic iron oxide particles with an aionic substance.

 8. The magnetic composite particles according to the above item 7, wherein the magnetic composite particles are silica, an ionic polymer, and a Z or ionic surfactant.

 9. The magnetic composite particle according to the above item 8, wherein the magnetic composite particle is a polymer having a carboxylic acid group, a sulfonic acid group, and a Z or phosphate group.

 10. The magnetic composite particle according to the above item 8, wherein the aionic surfactant is a surfactant having a carboxylic acid group, a sulfonic acid group and a Z or phosphate group.

[0015] 11. The magnetic composite particle according to any one of the above items 1 to 10, wherein the weight ratio of the magnetic particle to the chitosan layer is 100: 5-100: 300.

 12. The magnetic composite particle according to any one of the first to tenth, wherein the weight ratio of the magnetic particle to the chitosan layer is 100: 10 to 100: 90.

 13. The magnetic composite particle according to any one of the above items 1 to 12, wherein the thinnest portion of the chitosan layer has a thickness of 5 nm or more.

14. The magnetic composite particles according to any one of the above items 113, wherein the specific surface area of the magnetic composite particles is in the range of 100 m ² / g.

 15. The magnetic composite particle according to any one of the above items 1 to 14, wherein the coercive force of the magnetic composite particle is in the range of 0.80 to 15.92 kAZm (10 to 200 Oe).

16. Magnetic saturation magnetization of the composite particles 5- 80A'm ² / 1¾ magnetic composite particles according to any range of Aru the first one 15 of (5-80611111 / _8).

 17.a) a step of dissolving chitosan in water using an acid; b) a step of dispersing magnetic particles in the obtained chitosan solution; and c) a gradual dispersion of the resulting dispersion with a weakly alkaline base. A process for producing magnetic composite particles.

 18. The method for producing magnetic composite particles according to the seventeenth aspect, further comprising: d) adding a chitosan crosslinking agent into the system and stirring.

19. The method for producing magnetic composite particles according to the above item 17 or 18, further comprising the step of: e) making the neutral dispersion liquid obtained after the step c) alkaline quickly with a weak alkaline base. 20. The method for producing magnetic composite particles according to the above item 17-19, wherein the protective colloid is added into the system before the step c.

 The invention's effect

 As described above, the present invention provides magnetic composite particles having a chitosan layer on the surface of each of the magnetic particles by a specific method using a weak alkali base. As a result, it is possible to provide a composite material which can immobilize a physiologically active substance in a high yield without exposing the particles having the included magnetism, and which has good magnetic trapping properties. BEST MODE FOR CARRYING OUT THE INVENTION

 [0018] In the present invention, the magnetic composite particles having a chitosan layer on the surface of the particles having magnetism can be immobilized with a high yield of the physiologically active substance and have a high magnetic trapping property. It is effective. Here, the “having a chitosan layer on the surface of the magnetic particles” refers to a chitosan layer having a relatively uniform thickness without exposing the magnetic particles to the entire surface of the magnetic particles. Is covered, and refers to the form.

 That is, the surface of the magnetic composite particles of the present invention is coated with the chitosan layer, whereby the properties as chitosan, for example, the property of adsorbing various substances and fixing them are exhibited. In addition, since an amino group is introduced into the surface, it becomes possible to bond various functional groups and substances by a chemical reaction and immobilize them. Furthermore, since the chitosan has a uniform layered structure, the surface shape of the composite particles becomes closer to the surface shape of the magnetic particles, so that the surface area can be made larger than before, so that the adsorption, The yield of separation, purification, etc. can be increased. In addition, because of the uniform layered structure, chitosan has a feature that the thickness of the magnetic material is small and the internal magnetic material is hardly exposed.

 In the present invention, a more preferred form of the magnetic composite particles having a chitosan layer on the surface of the magnetic particles is a core-shell structure having magnetic particles as nuclei and chitosan as a shell. It is a magnetic composite particle. Basically, this form is small in size, close to the size of magnetic fine particles, in which one magnetic particle is included in one composite particle! can do.

As described above, attempts to produce composite particles of chitosan and magnetic fine particles have been made in the past. However, in these conventional methods, the magnetic fine particles as in the present invention are chitosan-coated. It is difficult to produce magnetic composite particles coated in a uniform layer.

 On the other hand, the present inventors, after intensive studies, disperse the magnetic fine particles in a chitosan solution in which chitosan is dissolved using an acid, and then gradually add a weak alkali base by a dropping method or the like. It was found out that a uniform layered structure was obtained when the pH was gradually increased by adding to the mixture. In addition, they found that a sufficiently uniform layered structure was formed even in the neutral range of pH 6-8. Furthermore, when the point used to dissolve the chitosan is neutralized (usually around pH 7.2), the chitosan is considered to crystallize, and the immobilization of chitosan on the surface of the magnetic particles becomes more difficult. Be certain. Therefore, it is desirable that the pH is ultimately higher than the above neutralization point, that is, a neutral and mono-alkaline state (usually pH 7.5 or more) closer to alkaline.

 In order to form a layered structure more uniformly, it is effective to extremely slow the rate of pH increase before and after the previously determined neutralization point in the operation of increasing the pH starting from the acidic condition. It is also effective to maintain the pH for a while after reaching a pH slightly lower than the previously determined neutralization point, and then raise the pH again to a pH higher than the neutralization point. is there.

 Conversely, after dispersing the magnetic fine particles in a weak alkaline aqueous base solution, a chitosan solution obtained by dissolving chitosan by making it acidic may be gradually added by a method such as a dropping method.

[0022] The above-mentioned prior art also discloses a technique for obtaining composite magnetic particles by adding a base. A strong base is added dropwise to a dispersion in which magnetic particles are dispersed by dissolving chitosan, or the above-described dispersion is performed. and added dropwise to solution in a strong base, thereby causing locally high _P H, and the deposition of abrupt chitosan accordingly. The deposited chitosan is difficult to re-dissolve, so that it forms a cluster of composite particles including the surrounding magnetic fine particles and does not form a uniform layered chitosan coating.

 On the other hand, if a weak alkali base is used, it is considered that the local high pH is suppressed, and no rapid chitosan precipitation occurs, and chitosan is gradually deposited on the surface of the magnetic particles. Such description is an important element of the present invention in the prior art.

As described above, to obtain a uniform layered structure, it is better to gradually increase the pH, but before and after the point of neutralizing the acid used to dissolve chitosan, chitosan is added. Coated composite Agglomeration of particles is likely to occur. When agglomeration occurs, a treatment for loosening the agglomeration may be performed, for example, by performing strong stirring or dispersion treatment, or by drying and disintegrating in a mortar.

 In addition, the present inventors have found that it is effective to shift from a neutral region to an alkaline region in a short time in order to suppress the above aggregation. Also in this case, when a strong base is used, on the contrary, a strong aggregate is easily formed. Therefore, it is preferable to use an unfavorably weak alkali base. In order to transfer to the alkaline region in a short time, it is effective to quickly add a large amount of this weak alkaline base and mix it, or to put the dispersion in a weak alkaline base. In particular, the latter method is preferable because aggregation of the composite particles is less likely to occur. On the other hand, when the chitosan solution is gradually added to the weak alkaline aqueous base solution in which the magnetic fine particles are dispersed, the above-described aggregation is effectively prevented.

 [0025] The weak alkali base used in the above method is not particularly limited, but those having a pH of 7.4-9.6 are preferable. If the pH is lower than this, chitosan will be sufficiently precipitated, and if the pH is higher than this, rapid precipitation of chitosan will occur and soon the composite particles will become huge, irregularly shaped, aggregated, etc. There is. pH 7.7-9.3 is more preferred.

 Representative weak alkaline bases include aqueous sodium hydrogen carbonate, ammonium dihydrogen phosphate solution, sodium dihydrogen phosphate solution, EDTA (ethylenediaminetetraacetic acid) 2Na solution, and ammonia gas. Further, among various buffers, those having a pH within the above range can be used. It is preferable to add the weakly alkaline base kneaded as an aqueous solution in order to suppress a local increase in pH. However, when the base is gaseous like ammonia, it is effective to blow the gas as it is.

[0026] The concentration of the base of the weak alkali varies depending on the type and pH of the base, and is generally preferably 1 N or less. If the concentration is higher than this, rapid chitosan precipitation is likely to occur. It is more preferably 0.7N or less. In particularly high _P H, it is often preferred better to suppress the concentration low. For example, in many cases, it is preferable to set the pH to 0.4N or less for a base having a pH of 9-9.5, and 0.2N or less for a base exceeding pH 9.5.

On the other hand, the lower limit of the concentration of the weak alkali base is determined by process restrictions. The lower the concentration, the easier it is to produce a uniform chitosan.The amount of base solution added increases, the lower the concentration of chitosan and magnetic particles, the lower the yield, and the lower the concentration. This is disadvantageous in cost because the amount of the drug increases and a large container is required. In general, 0.001N or more is preferable, and 0.01N or more is more preferable.

 [0027] The chitosan in the present invention is not particularly limited, and various types obtained by deacetylating chitin can be used. Various deacetylated chitosans are also commercially available, and an appropriate one may be selected and used according to the application. In addition, if necessary, chitosan to which other substances are bound may be used. However, in order to obtain a uniform layered structure using the above-mentioned method, it is usually necessary to be alkaline and insoluble in water.

 [0028] Further, since chitosan is generally insoluble in neutral or alkaline water, the covered chitosan can be used in a wide range without cross-linking. However, when used under acidic conditions, or when adsorption, immobilization, modification, etc. are performed to increase the solubility of chitosan, it is preferable to crosslink so that chitosan does not elute.

 The cross-linking reaction is preferably performed after the chitosan layer has been formed to some extent or more. If the cross-linking reaction is carried out in a state where a large amount of chitosan is dissolved, the whole dispersion may be gelled. Specifically, it is preferable to carry out the crosslinking reaction at pH 6 or more.

 [0029] If the pH is 6 or more, the crosslinking reaction may be performed in any of acidic, neutral, and alkaline states. When a crosslinking reaction is performed, crosslinking is likely to occur around the chitosan layer surface. These may be selected according to the application.

 [0030] The method for crosslinking chitosan is not particularly limited, but it is effective to use a crosslinking agent that reacts with two or more of amino groups, hydroxyl groups, and acetoamide groups contained in chitosan. Representative functional groups that can react with these functional groups include aldehyde groups, epoxy groups, isocyanate groups, carboxyl groups, acid anhydride groups, acid chloride groups, and the like. It is effective to use a crosslinking agent having two or more functional groups selected from the group. In particular, aldehyde groups, epoxy groups, and isocyanate groups are preferably used because they can undergo a crosslinking reaction under relatively mild conditions. The two or more functional groups in the cross-linking agent may be the same or different, but in the case of different types, a combination that does not easily react in the molecule is desirable. The crosslinking agent may be a low molecular compound or a high molecular compound.

[0031] In the crosslinking reaction, if necessary, a catalyst, a reactive group activator, a buffer, etc. may be used. May be. For example, a tertiary amine is used for the reaction between the amino group and the epoxy group, and a dehydrating agent or carbodiimide is preferably used for the reaction between the amino group and the carboxyl group. Further, the crosslinking reaction may be performed by heating to a temperature required for the reaction.

 [0032] The amount of the cross-linking agent to be added can be selected according to the use, but since chitosan is usually of a high molecular weight, it is often sufficient to add 0.1% by weight or more to chitosan. If the strength or low swelling of chitosan is required, it is advisable to increase the amount of the crosslinking agent added. It is preferable to add 1% by weight or more for more reliable cross-linking, and it is preferable to add 3% by weight or more for suppressing elution of chitosan into an acidic substance.

 On the other hand, if a functional group such as an amino group is required, all of the functional groups must not undergo a crosslinking reaction, and the amount of the crosslinking agent added must be suppressed so that functional groups such as an amino group remain. Since the residual ratio of the functional group varies depending on the reactivity, reaction conditions, particle size, ratio of particles having chitosan Z magnetism, etc., the amount of the crosslinking agent to be added is appropriately determined in consideration of these conditions.

 In order to increase the crosslink density and increase the amount of amino groups, it is also effective not to hydrolyze! / After performing the crosslinking reaction, hydrolyzing the acetoamide groups is also effective. When the amount of amino groups is particularly increased, chitosan or chitin having a low degree of deacetylation may be used. If the degree of deacetylation is too low, it is difficult to deposit chitosan due to neutrality and alkalinity, so that the degree of deacetylation is preferably 70% or more.

 The prior art is one of the unique techniques of the present invention that mentions a method of performing deacetylation after forming composite particles.

 The method of deacetylation is not particularly limited, and conventionally known methods such as alkali treatment and enzyme reaction can be used. For example, the alkali treatment may be performed by heating to 80 to 120 ° C in a concentrated alkaline solution such as 45% NaOH and treating for 45 hours. In this case, it is important that the cross-linking is not broken by the deacetylation reaction. It is also effective to carry out a crosslinking reaction by protecting the amino group with a protecting group such as a metal ion complex and a Schiff base, and to remove the protecting group after crosslinking.

[0034] The amount of the remaining amino groups can be determined by titration. A typical titration method is as follows. Put the magnetic composite particles in ethanol, and rotate the stirrer tip at a rotation speed at which the particles are sufficiently stirred. Wait until the potential stabilizes, and then use it as a titration reagent. Potentiometric titration is performed using a hydrochloric acid solution in ethanol. For titration, inject a small amount of titrant, wait until the potential stabilizes, and perform intermittent titration with the next titrant, or very slowly. If an acid or base is added during production for pH adjustment, reaction promotion, etc., it is important to purify by washing well or by performing ion exchange resin treatment.

 However, according to this technique, uncrosslinked chitosan chains may be eluted, so that even an internal amino group which does not exhibit an effect during actual use may be detected. It is desirable to apply to fully crosslinked magnetic composite particles. In addition, it is important that the crosslinking bond is not cleaved by hydrochloric acid. Further, another acid may be selected.

 [0035] The amount of residual amino groups in the magnetic composite particles obtained as described above is preferably 0: L mol or more per lg of the magnetic composite particles. If the amount is less than this, the efficiency of immobilizing the physiologically active substance will decrease. More preferably, it is 0.001 mmol or more, more preferably 0.005 mmol or more per lg of the magnetic composite particles. On the other hand, the upper limit of the residual amino group is usually 5 mmol Zg or less, since the amount of the amino group of chitosan itself is about 5 mmol Zg and magnetic particles are contained.

 [0036] In the present invention, the force of using magnetic particles as nuclei allows the composite particles to have magnetism, and after the bioactive substance is immobilized on the magnetic particles, it can be easily applied using a magnet or the like. It has the characteristic that it can be collected in Magnetic particles are not particularly limited! ヽ However, ferromagnetic iron oxide particles have excellent magnetic properties and can produce ultra-fine particles, so they are suitable for use where fine particles are particularly desirable. Is done.

 Ferromagnetic oxide particles include magnetite (Fe 2 O 3) particles and maghemite (γ-Fe 2 O 3

 3 4 2 3

) Particles, Magnetite-Magnet Intermediate Oxide Iron Particles, Gamma Hematite-to-Alpha Matite Intermediate Oxide Iron Particles, Manganese Zinc Ferrite HMnZnFe O) Particles, Rare Earth Iron Gas

 twenty four

 At least one selected from the group consisting of one net particle and bismuth-substituted rare earth iron garnet particles is preferred. In particular, magnetite particles are optimal because they have good magnetic field sensitivity when collected by a magnet with a large saturation magnetization.

Since chitosan is a cationic substance, in the step of coating with chitosan, if the surface of the magnetic particles has an auronic property, the particles are uniformly coated because they come close to each other. It becomes easier to obtain a cover.

 Accordingly, among the above ferromagnetic oxide particles, particles having a strong ionization property such as mag hematite can easily obtain a uniform coating without any particular surface treatment. On the other hand, uniform coating can be easily obtained on strongly cationic particles by applying an iron-based substance and performing a coating process with a strong chitosan.

 Examples of the a-on substance to be deposited on the ferromagnetic iron oxide particles in advance include silica, an a-on polymer, and a Z or a-on surfactant. Examples of the a-on polymer include polymers having a carboxylic acid group, a sulfonic acid group and z or a phosphoric acid group. Specific examples include (meth) acrylic acid and (meth) atalyloyloxy. Synthetic polymers obtained by copolymerizing shetyl succinic acid, maleic acid, acrylamido-2-methylpropane sulfonic acid, styrene sulfonic acid, 2- (meth) atalyloyloxyshetyl acid phosphate, carboxymethyl cellulose, alginic acid, etc. Semi-synthetic or natural polymers are included. Examples of the aionic surfactant include a surfactant having a carboxylic acid group, a sulfonic acid group and a Z or phosphate group, and specific examples thereof include stearic acid, myristic acid, and oleic acid. Acid, dodecylbenzenesulfonic acid, and the like.

 [0037] The weight ratio of ferromagnetic iron oxide particles to chitosan is preferably from 100: 5 to 100: 300. If the ratio of chitosan is higher than 100: 300, the ratio of magnetic particles will be lower and the magnetic properties of the composite particles will not only be reduced, but more than one magnetic particle will be encapsulated in one composite particle. The risk increases. It is more preferable that the amount of chitosan is less than 100: 200, and it is even more preferable that the amount of chitosan is less than 100: 90. With a chitosan content of 100: 40 or less, a sufficiently thick chitosan layer can be obtained for many applications.

 On the other hand, if the ratio of chitosan is lower than 100: 5, it becomes difficult to cover the whole of the magnetic particles. When the ratio is 100: 5, the magnetic particles having a still thinner coating may be easily exposed, so that it is more preferable that the ratio of chitosan is higher than 100: 10. Further, it is most preferable that the amount of chitosan is large in a ratio of 100: 15 or more, which can provide a chitosan layer having a sufficient thickness with respect to the particle diameter of the magnetic particles.

[0038] In order to prevent the magnetic particles inside from being exposed, the chitosan layer must have a sufficient thickness. Even if a layered structure is formed, if the layer thickness is small, the thickness of the chitosan layer The internal magnetic particles may be exposed due to mechanical effects such as wobble, irregularities on the surface of the magnetic particles, and rubbing or collision. Considering the factors affecting these exposures, it is desirable that the thickness of the chitosan layer be 5 nm or more at the thin part. 1

Onm or more is more preferable 20 nm or more is more preferable.

 The thickness of the thin portion of the chitosan layer was about

From the 50 particles, select the thin part of the chitosan layer and measure the thickness, and from the thinnest point

Find the average of 10 points!

[0039] The thickness of the chitosan layer is substantially determined by the particle size of the magnetic particles and the weight ratio between the ferromagnetic oxide particles and chitosan. Therefore, to obtain a desired thickness of the chitosan layer, The weight ratio of ferromagnetic iron oxide particles to chitosan should be adjusted to the magnetic particles used. On the other hand, when the thickness of the chitosan layer is increased, the ratio of the magnetic particles to the entire composite particles is reduced, so that the magnetic characteristics of the composite particles are weakened. Therefore, it is necessary to keep the thickness at the upper limit of the weight ratio between the ferromagnetic oxide particles and chitosan.

The specific surface area of the [0040] magnetic composite particles, when in the range of 0. 1- 100m ² / g, since the fixing I匕量of a physiologically active substance is increased, it is preferably used. When the specific surface area is smaller than the above range, the amount of the bioactive substance that can be fixed per unit weight of the magnetic composite particles decreases, and a large amount of the magnetic composite particles is required to obtain the effect of immobilizing the bioactive substance. Is not good. More preferably lm ² Zg or more, even more preferably 3 m ² Zg or more.

The specific surface area is preferably higher as the amount of the immobilized physiologically active substance becomes larger, but when the specific surface area is increased, the particles are usually smaller and the collecting property by the magnetic field is lower. There are many pores that are difficult for bioactive substances to enter! Therefore, the specific surface area is preferably 100 m ² Zg or less. Further, 5 Om ² Zg or less is more preferable, and 20 m ² Zg or less is further preferable.

[0041] The particle size of the magnetic composite particles after forming a uniform layered structure is such that the average particle size is in the range of 0.01- when fine particles such as ferromagnetic oxide particles are used as nuclei. Preferably it is. If the average particle size is smaller than the above range, the trapping property by a magnetic field tends to be low. Further, if the average particle size is larger than the above range, the particles Even if it disperses, it will settle down immediately. More preferably, the average particle size is between 0.03 and 2 m, more preferably between 0.1 and 1 m. The particle size of the magnetic composite particles can be determined from the average particle size by measuring about 50 particle sizes on a transmission electron micrograph.

 Next, regarding the coercive force of the magnetic composite particles, in general, when the coercive force increases, the cohesive force between the magnetic particles increases, and the dispersibility decreases. As a result, the number of active sites to be bound to the physiologically active substance decreases, and the efficiency of immobilizing the physiologically active substance tends to decrease.

 In the magnetic composite particles of the present invention, since the chitosan coating is formed on each of the magnetic particles, the coercive force of the magnetic composite particles is substantially determined by the coercive force of the magnetic particles themselves. It is decided.

 The present inventors have conducted intensive studies on the optimum coercive force range that does not affect the immobilization characteristics of the physiologically active substance. As a result, the range of 0.80 to 15.92 kAZm (10 to 200 Oersted) was obtained. Then, they found that there was no practical problem.

 When the coercive force is larger than 15.92 kAZm, the dispersibility of the magnetic composite particles decreases, but it was found that there is no practical problem if the coercive force is less than 15.92 kAZm. There is no particular problem for the low coercive force, but when the ferromagnetic oxidized iron particles are used as nuclei, the particle size of the ferromagnetic oxidized iron particles is increased to be lower than 0.80 kAZm. It is necessary to make ferromagnetic iron oxide particles into a shape or structure that is not suitable for the purpose of the present invention.

Next, the saturation magnetization of the magnetic composite particles is determined by the saturation magnetization of the magnetic particles and the amount of the chitosan layer to be formed, and is preferably 5 A-m ² Zkg (5 emuZg) or more. If the saturation magnetic drier is smaller than 5 A'm ² Zkg, collection by a magnet tends to be difficult. When the saturation magnetic field is 20 A'm ² / kg or more, the trapping property is high, so that it is more preferable 40 A-m ² / kg or more. The upper limit is not particularly limited, but is limited by the saturation magnetization of the magnetic particles. For example, when magnetite is used, the upper limit is usually 80 A'm ² / kg (80 emu / g) or less.

[0045] As the magnetic particles in the present invention, various shapes such as needle-like, plate-like, spherical, granular, elliptical, and cubic shapes can be used, but a coating having a uniform thickness can be easily formed. spherical, Elliptical and granular shapes are particularly preferred. Here, the term “spherical” refers to a shape whose aspect ratio (the ratio of the maximum length to the minimum length when measured in all directions) is within the range of 1.0-1.2, and “spherical”. "" Means a shape with an aspect ratio in the range of 1.2-1.5. In addition, the term “granular” refers to a particle having a uniform length in all directions, such as a sphere, or a particle having a length in only one direction, such as an ellipse. However, as a whole, particles have no particular anisotropy and refer to particles.

[0046] In the present invention, the method for producing ferromagnetic iron oxide particles used as the particles having magnetism is not particularly limited. As an example, as a method for producing magnetite particles, a synthesis method using an oxidation reaction of an iron salt in an aqueous solution will be described below.

 First, a NaOH aqueous solution was added to a divalent aqueous Fe ion solution in which ferrous sulfate (FeSO6 ·) was dissolved.

 4 2

 The solution is added dropwise to precipitate ferrous hydroxide [Fe (OH)]. This suspension of ferrous hydroxide

 2

 The pH of the solution is adjusted to 910, and air is blown into the solution to grow the magnetite particles. If the pH is lower than the above range, the precipitation of magnetite will be slow, and if it is higher than the above range, goethite (a FeOOH) will be easily generated.

 [0047] The air blowing speed and the suspension holding temperature greatly affect the particle size of the magnetite particles. It is advisable to adjust the air blowing speed to 100-400 liters Z-hour and the suspension holding temperature to 50-90 ° C. If the air blowing speed is high, the crystal growth of magnetite will be faster and the particle size will be smaller. If the air blowing speed is too low or too high, substances other than magnetite are liable to coexist. As the holding temperature increases, magnetite crystal grows more easily and the particle size increases. If the retention temperature is too low, goethite (α-FeOOH) particles are likely to be generated.

 By such a method, magnetite particles having an average particle size of 0.01-0.5 m can be synthesized. The above average particle size is obtained by measuring the size of 50 particles on a scanning electron micrograph and determining the average force.

 [0048] In the present invention, the method for producing magnetic composite particles having a uniform layered structure of chitosan on the surface of particles having magnetic properties such as ferromagnetic oxide particles such as ferromagnetic oxide particles is particularly limited. Although not specified, a typical production method is as follows.

Since chitosan is usually in a solid state, after chitosan is poured into water, while stirring, The ability to add hydrochloric acid, acetic acid, formic acid, lactic acid, etc., and dissolve chitosan by adding chitosan to a previously prepared aqueous solution of these acids and stirring. If insoluble matter remains, it is desirable to filter. The amount of chitosan is preferably 1% by weight or less for ordinary chitosan, which is desirably an amount such that the solution viscosity after dissolution is sufficiently stirrable. Particles having a desired ratio of magnetism to the chitosan, for example, magnetite particles synthesized by the above-described method, are added and sufficiently dispersed to prepare a suspension.

 [0049] Next, while stirring this suspension, an aqueous solution of sodium hydrogen carbonate, a solution of ammonium dihydrogen phosphate, and a solution of diphosphate having a pH of 0.001 to 1N and a pH of 7.4 to 9.6 were prepared. Sodium hydrogen solution, EDTA (ethylenediaminetetraacetic acid) '2Na solution, or the force to drop a weakly alkaline base such as a buffer solution adjusted to the above pH range, or by blowing in ammonia gas, gradually adjust the pH of the suspension. Raise the pH and neutralize the acid used to dissolve chitosan from pH 6 (usually around pH 7.2), and keep it for a while. As a result, chitosan is precipitated on the surface of the magnetic particles.

 Here, if necessary, cross-linking of chitosan is performed. As a crosslinking method, for example, a diluent such as datalaldehyde or a commercially available epoxy crosslinking agent may be added to the uncrosslinked composite particle suspension and stirred. At this time, if necessary, heating, addition of a catalyst, and the like may be performed. If the crosslinking reaction is slow, or if the crosslinking reaction is difficult to proceed at acidic or room temperature, the crosslinking agent may be added at the previous stage.

 Further, a protective colloid may be added into the system before the reaction for precipitating chitosan. Thereby, the dispersion stability of the magnetic particles and the particles coated with chitosan is improved, the aggregation of the particles is suppressed, and the uniformity of the chitosan coating is easily obtained. Examples of the protective colloid include polyvinyl phenol renore, methinoreseno reloose, hydroxyethino reseno reloose, hydroxypino pinole reloose, and the like.

[0050] Further, in order to more securely fix chitosan on the surface of the magnetic particles, the dispersion liquid is neutralized with a weak alkali base to neutralize the acid used for dissolving chitosan. Adjust the pH to a higher level (usually around pH 7.2), that is, to a more alkaline-neutral, mono-alkaline condition (usually pH 7.5 or higher).

At this time, the aggregation of the composite particles is suppressed by quickly adjusting the neutrality to alkaline. be able to. It is particularly effective to drop the dispersion liquid in a weak alkali base. At this stage, the chitosan may be crosslinked. The crosslinking method is as described above.

 The uncrosslinked or crosslinked magnetic composite particles thus obtained are subjected to washing, for example, to remove unadhered substances and unreacted substances, and then, if necessary, are subjected to concentration, drying, solvent replacement, mixing of additives, and crushing. Thereby, the magnetic composite particles targeted by the present invention are obtained.

 [0051] The characteristics of the magnetic composite particles of the present invention are summarized as follows.

 (1) A chitosan coating layer having a uniform and sufficient thickness is provided on the surface of magnetic particles. As a result, the surface area can be increased, and even if the ratio of particles having magnetic properties inside is increased, the exposure can be suppressed.

 (2) In order to obtain the form shown in the above (1), magnetic particles are dispersed in a chitosan solution dissolved using an acid, and gradually neutralized using a weak alkali base.

 (3) Regarding the pH adjustment with a weak alkali base, in order to more securely fix chitosan on the surface of the magnetic particles, the acid used for finally dissolving the chitosan is neutralized. Make the pH higher than the summation point (usually around pH 7.2), that is, more alkaline-neutral monoalkaline (usually pH 7.5 or higher).

 (4) In order to suppress the aggregation of the composite particles when the pH increases, it is effective to make the alkali particles promptly alkaline with a weak alkali base. It is particularly effective to drop the dispersion onto a weakly alkaline base.

 (5) In the case of cross-linking, if a cross-linking agent is added and reacted under neutral conditions, cross-linking of the chitosan layer more even inside can be achieved.

 Hereinafter, the present invention will be described specifically with reference to Examples. However, the present invention is not limited by the following Examples, but may be appropriately modified within a range that can conform to the gist of the preceding and following descriptions. Of course, it is also possible to carry out the present invention, and all of them are included in the technical scope of the present invention.

 Example 1

<Synthesis of magnetite particles>

 lOOg ferrous sulfate (FeSO · 7Η Ο) was dissolved in 1, OOOcc pure water. This sulfuric acid

 4 2

28.8 g of sodium hydroxide was dissolved in 500 cc of pure water so as to be equimolar to one iron. . Next, an aqueous solution of sodium hydroxide was added dropwise over 1 hour while stirring the aqueous ferrous sulfate solution to form a precipitate of ferrous hydroxide. After completion of the dropwise addition, the temperature of the suspension containing the precipitate of ferrous hydroxide was increased to 75 ° C while stirring. After the temperature of the suspension reached 75 ° C, it was oxidized for 8 hours at a rate of 250 liters Z hour while blowing air using an air pump to produce magnetite particles.

The magnetite particles thus obtained were sufficiently washed with pure water, filtered, and dried by heating at 50 ° C. under reduced pressure. The magnetite particles are almost spherical, the average particle size is about 0.23 μm, the specific surface area is 7.Om ^{2, the} coercive force is 5.2 kA / m, and the saturation magnetization is 82.8 A-m ² Zkg. The particle size of magnetite particles was determined from the average particle size of about 50 particles measured on a transmission electron microscope.

[0054] Kuchitosan Coating Layer Forming Treatment>

 1,5 g of chitosan is dispersed in 1,000 g of pure water, and 5 g of acetic acid is gradually added dropwise to obtain chitosan (“PSH-80” manufactured by Yaizu Suisan Chemical Co., Ltd., deacetylation degree of 80% or more). Dissolved. If there were any insolubles, they were removed by filtration.

 To 20 lg of this chitosan solution, 20 g of the above magnetite particles were mixed and ultrasonically dispersed. While stirring the dispersion, a 0.5N aqueous sodium hydrogen carbonate solution was gradually added dropwise to adjust the pH to 7.1, and then stirring was continued for 1 hour. Next, a 0.1N aqueous sodium hydrogen carbonate solution was added dropwise to the mixture to adjust the pH to about 8, and the mixture was further stirred for 1 hour.

 The magnetic composite particles thus obtained are sufficiently washed with pure water, filtered, dried by heating under reduced pressure at 40 ° C, and crushed in a mortar to form a uniform coating layer of chitosan. The obtained magnetic composite particles were obtained.

 Example 2

 [0055] By performing the same treatment as in Example 1 except that the amount of the magnetite particles was changed from 20 g to 9 g in the formation processing of the chitosan coating layer, the magnetic layer having a uniform chitosan coating layer was formed. Composite particles were obtained.

 Example 3

[0056] A uniform coating layer of chitosan was formed by performing the same processing as in Example 1 except that the amount of magnetite particles was changed from 20 g to 5 g in the process of forming the coating layer of chitosan. The resulting magnetic composite particles were obtained.

 Example 4

 [0057] A uniform coating layer of chitosan was formed by performing the same treatment as in Example 1 except that the amount of magnetite particles was changed from 20 g to 1.2 g in the formation process of the chitosan coating layer. Magnetic composite particles were obtained.

 Example 5

 [0058] A uniform coating layer of chitosan was formed by performing the same process as in Example 1 except that the amount of magnetite particles was changed from 20 g to 0.5 g in the formation process of the chitosan coating layer. Magnetic composite particles were obtained.

 Example 6

 [0059] A uniform coating layer of chitosan was formed by performing the same processing as in Example 1 except that the amount of magnetite particles was changed from 20 g to 0.25 g in the formation process of the chitosan coating layer. Magnetic composite particles were obtained.

 Example 7

 [0060] In the process of forming the coating layer of chitosan, the amount of magnetite particles was changed from 20 g to 9 g, and before washing with water, a 1% dilute aqueous solution of dartal aldehyde with respect to chitosan was stirred in the dispersion. Then, the same treatment as in Example 1 was performed except that the reaction was carried out at room temperature for 1 day, to obtain magnetic composite particles having a uniform coating layer of chitosan.

 Example 8

 [0061] In the treatment for forming the coating layer of chitosan, the amount of the magnetite particles was changed from 20 g to 5 g, and the dispersion after stirring for 1 hour at pH 7.1 was stirred in 100 ml of a 0.5N aqueous sodium hydrogen carbonate solution. Then, magnetic composite particles having a uniform coating layer of chitosan were obtained in the same manner as in Example 1, except that the mixture was further stirred for 1 hour, and then treated after water washing. Example 9

[0062] The magnetite particles obtained in the synthesis of the magnetite particles of Example 1 were subjected to heat treatment at 200 ° C for 2 hours to obtain maghemite particles having an average particle size of 0.23 m. These particles have a specific surface area of 6.9 m ² Zg, a coercive force of 5.2 kAZm, and a saturation magnetization of 79.3 A-mV kg.

 Magnetic composite particles having a uniform coating layer of chitosan were obtained by performing the same treatment as in Example 2 except that the same weight was used instead of the magnetite particles.

 Example 10

 The magnetite particles obtained by the synthesis of the magnetite particles were subjected to a silica coating treatment of 10% by weight (prepared value) by a sol-gel method according to a conventional method to obtain silica-coated magnetite particles.

 Magnetic composite particles having a uniform coating layer of chitosan were obtained by performing the same treatment as in Example 2 except that the same weight was used instead of the magnetite particles.

 Example 11

 [0064] The magnetite particles obtained in the synthesis of the magnetite particles were stirred in an aqueous solution in which 2% by weight of polyacrylic acid (PAA) was dissolved with respect to the magnetite, to obtain magnetite particles coated with polyacrylic acid. Was.

 Magnetic composite particles having a uniform coating layer of chitosan were obtained by performing the same treatment as in Example 2 except that the same weight was used instead of the magnetite particles.

 Example 12

 [0065] The magnetite particles obtained by synthesizing the magnetite particles were stirred in an aqueous solution in which 2% by weight of oleic acid (OA) was dissolved with respect to the magnetite to obtain oleic acid-coated magnetite particles.

 Magnetic composite particles having a uniform coating layer of chitosan were obtained by performing the same treatment as in Example 2 except that the same weight was used instead of the magnetite particles.

 Example 13

 [0066] In the process of forming the coating layer of chitosan, a uniform coating layer of chitosan was obtained by performing the same treatment as in Example 2 except that 201 g of the chitosan solution was dissolved with 1 g of polyvinyl alcohol PVA-117. The formed magnetic composite particles were obtained.

 Example 14

[0067] Magnetite having an average particle size of about 0.02 μm was adjusted by adjusting the conditions for synthesizing magnetite particles. Particles were obtained. The magnetite particles were almost spherical, the specific surface area was 47 m ² Zg, the coercive force was 5. lkAZg, and the saturation magnetization was 73.7 A'm ² Zkg. The magnetite particles were subjected to a silica coating treatment of 10% by weight (prepared value) by a conventional sol-gel method to obtain magnetite particles coated with silica having an average particle size of about 0.02 m.

 0.25 g of the magnetite particles were mixed with 201 g of the chitosan solution, followed by ultrasonic dispersion. This dispersion was emulsified and dispersed in hexane, and then the emulsified dispersion was poured into methanol to obtain magnetic composite particles having a uniform coating layer of chitosan.

 Regarding the magnetic composite particles obtained in Example 18 above, the amounts of the magnetite particles, chitosan and water used in the treatment for forming the chitosan coating layer, the weight ratio of the magnetite particles Z chitosan, and the presence or absence of crosslinking Are summarized in Table 1.

 [Table 1]

 Comparative Example 1

In the same manner as in Example 1, a magnetite particle-dispersed aqueous solution of chitosan was prepared at a ratio of 1.35 g of magnetite particles, 0.15 g of chitosan, and 200 g of pure water (magnetic particles / chitosan = 100Zl l. 1). Then, 10 ml of an aqueous sodium hydroxide solution (pH = 13.2) was gradually added dropwise thereto, and 0.086 g of epichlorohydrin was further added. The mixture was heated and refluxed for 2 hours. The obtained magnetic composite particles were sufficiently washed with pure water, filtered, and dried by heating under reduced pressure at 40 ° C to obtain chitosan magnetic composite particles.

[0071] Comparative Example 2

 Chitosan magnetic composite particles were obtained in the same manner as in Comparative Example 1, except that 10 ml of a 1N aqueous sodium hydroxide solution was changed to 10 ml of a 1N aqueous ammonia solution (pH = 11.8).

[0072] Comparative Example 3

 Chitosan magnetic composite particles were obtained in the same manner as in Comparative Example 1, except that 10 ml of a 1N aqueous sodium hydroxide solution was changed to 10 ml of a 1N aqueous sodium carbonate solution (pH = 10.0).

[0073] Comparative Example 4

 Chitosan 100 g was added to 1,000 ml of an ethanol solution of hydrochloric acid (concentrated hydrochloric acid Z ethanol = 1Z9), and the mixture was refluxed at 80 ° C. for 2 hours with stirring. After cooling and filtering, the mixture was washed with 1,000 ml of 90% by volume of ethanol, and filtered again.

 The chitosan particles collected by filtration were added to 1,000 ml of an ethanol solution of sodium hydroxide (4N sodium hydroxide, Zethanol = 1Z9) and stirred at room temperature for 4 hours. In the same manner as described above, 1,000 ml of 90% by volume ethanol was washed, filtered, and dried under reduced pressure for 12 hours to obtain low molecular weight chitosan.

[0074] 20 g of this depolymerized chitosan was dissolved in 1,000 ml of 0.1 N hydrochloric acid. To this, 2.78 g of the magnetite particles obtained in Example 1 was added (magnetic particles Z chitosan = 100/721), and ultrasonically dispersed. This dispersion was granulated by spray drying. Spray drying was performed using a spray dryer manufactured by Sakamoto Giken Co., Ltd., with a hot air temperature of 180 with an atomizer-disk system. C, exhaust air temperature: 85 ° C, stock solution supply speed: 130 mlZ, disk rotation speed: 13, OOOrpm, and sprayed to obtain chitosan magnetic composite particles.

 The chitosan-magnetic composite particles 2.Og thus obtained were added to 200 ml of 90 vol% ethanol in which 0.4 g of sodium hydroxide was dissolved, and ultrasonically dispersed. To this, 0.67 ml of chloromethyloxylan was added, and the mixture was refluxed at 80 ° C. for 2 hours with stirring.

After cooling and filtering, wash with 200 ml of 90% by volume ethanol and dry under reduced pressure for 12 hours. Thus, crosslinked chitosan magnetic composite particles were obtained.

[0075] Comparative Example 5

 0.3 g of the magnetite particles obtained in Example 1 was added to 50 ml of water and ultrasonically dispersed. To this, a chitosan solution in which 0.03 g of chitosan was dissolved in 4.5 ml of 1% acetic acid was added. The dispersion was again ultrasonically dispersed, and the magnetic composite particles were collected by a magnetic field. The supernatant was filtered and dried under reduced pressure for 12 hours to obtain chitosan magnetic composite particles.

[0076] The average particle size, coercive force, saturation magnetization, specific surface area, magnetic field trapping property, coating property of each composite particle obtained in Examples 1-114 and Comparative Examples 1-1-5 were determined by the following methods. The uniformity and the thickness of the thin part of the chitosan layer were measured and evaluated. The results were as shown in Table 2.o

 <Measurement of Average Particle Size>

 Scanning electron micrographs of the composite particles were taken, the size of 50 particles was measured on this photograph, and the average value was determined.

<Measurement of coercive force and saturation magnetization>

 The coercive force and saturation magnetization of the composite particles were measured using a vibrating sample magnetometer (manufactured by Toei Industry Co., Ltd.). Saturation magnetization was determined from the amount of magnetization when a magnetic field of 797 kAZm (10 kOe) was applied.

<Measurement of Specific Surface Area>

 The specific surface area was measured using a specific surface area / pore distribution measuring device (“SA3100” manufactured by Coulter Inc.).

<Evaluation of magnetic field trapping property>

 After adding 0.5 g of the composite particles to 1.5 g of water and stirring well, the ferrite magnet is brought into contact with the side of the container, and the time until the composite particles are collected by the magnet portion and the liquid becomes almost transparent is measured. Was.

 Those that became transparent within 10 seconds were rated as ◎, those that took 10-30 seconds to become transparent, Δ that took 30 seconds and 12 minutes to become transparent, and those that took more time were rated X.

<Evaluation of coating uniformity>

Observe the composite particles with a transmission electron microscope (or scanning transmission electron microscope), and The chitosan coating on the surface of the child was evaluated to determine whether the force was uniform in thickness and whether the thickness was sufficient. If the coating thickness was almost uniform and the thickness was 20 nm or more, ◎, those that were almost unsatisfied, X were evaluated, and those between them were evaluated in two stages, 〇 and △ .

 <Measurement of thickness of thin portion of chitosan layer>

 In the transmission electron microscope (or scanning transmission electron microscope) photograph of the composite particles, the neutral part of about 50 particles is selected, the thickness of the thin part of the chitosan layer is measured, and the average of the ten points from the thinnest point is calculated. Was.

[Table 2]

From the results in Table 2 above, it was found that each magnetic composite particle of Example 1-14 of the present invention, particularly each magnetic composite particle of Example 1-13, in which a chitosan layer was formed on the surface of a magnetic particle by adding a weak alkali base. The composite particles are smaller than the composite particles of Comparative Examples 14 to 14 in which a plurality of magnetic particles are coated with chitosan by dropping or spray drying of a strong alkali base. It can be seen that the specific surface area can be increased.

 Also, the magnetic composite particles of Example 114, particularly of Example 113, were smaller than the composite particles of Comparative Example 14 and the composite particles of Comparative Example 5 coated with chitosan with an acidic solution. Tosan forms a layered structure and has a structure in which inclusions (magnetic particles) with high uniformity of coating thickness and high coating thickness are difficult to be exposed. That's all.

Next, an enzyme immobilization test was performed on these composite particles.

 First, a fixed amount of the composite particles was dispersed in water, a certain amount of peroxidase as an enzyme was added to the dispersion, and the mixture was stirred to immobilize peroxidase on the composite particles. As a method for measuring the amount and activity of the immobilized enzyme, a color reaction of TOOS-4 AA system was used. This color reaction was carried out using oxygen generated by the reduction of hydrogen peroxide and peroxidase catalyzed by TOOS. The reaction of [N-ethyl-N- (2-hydroxy-3sulfopropyl) -3-methyladiline] and 4-AA (4-aminoantipyrine) produces a dye that absorbs at a wavelength of 546 nm. Is a method known to those skilled in the art.

First, 0.1 ml of an aqueous solution containing 0.9% by weight of hydrogen peroxide was added to a reaction solution [50 mM Tris-HCl buffer (pH 7.5), 0.6 mM 'TOOSm 0.5 mM 4-AA] was added to the mixture, and the mixture was stirred to prepare a measurement solution.

 Next, lOOmg of composite particles having peroxidase immobilized thereon was added to the measurement solution, and the mixture was heated at 37 ° C. With respect to this solution, the absorbance (OD: 546 nm) for 5 minutes immediately after the addition of the composite particles was measured at an interval of 30 seconds using an absorptiometer, and the dyes produced by modifying the hydrogen peroxide in the above samples were compared.

 As a result, in the magnetic composite particles of Examples 114, although the enzyme activity of the composite particles of Example 1 was slightly lower, a larger amount of enzyme was immobilized than the composite particles of Comparative Example 1. It was also found that the immobilized enzyme exhibited high activity. Industrial applicability

[0087] The magnetic composite particles of the present invention can be used for immobilizing a physiologically active substance.

For example, diagnostic drug carriers, bacterial separation carriers, nucleic acid separation and purification carriers, protein purification carriers, It can be used as an immobilized enzyme carrier, an antibody-immobilized carrier, and the like. Also, due to its properties, it can be widely used for contrast agents and hyperthermia.

Claims

The scope of the claims  [I] Magnetic composite particles having a chitosan layer on the surface of magnetic particles and having an average particle size in the range of 0.01 to 4 μm.  [2] The magnetic composite particle according to [1], wherein the magnetic composite particle has a core-shell structure in which the magnetic particle is a core and the chitosan layer is a shell. [3] The magnetic composite particle according to claim 1 or 2, wherein the chitosan layer is crosslinked. 4. The magnetic composite particle according to claim 3, wherein a crosslinking agent having two or more functional groups selected from the group consisting of an aldehyde group, an epoxy group, and an isocyanate group is used for crosslinking of the chitosan layer. [5] The magnetic composite particles according to any one of claims 14 to 14, wherein the particles having magnetism are ferromagnetic oxide particles.  [6] Ferromagnetic oxide particles Magnetite particles, maghemite particles, magnetite-maghemite intermediate oxide particles, gamma hematite alpha hematite intermediate oxide particles, manganese zinc ferrite particles 6. The magnetic composite particles according to claim 5, wherein the magnetic composite particles are at least one selected from the group consisting of rare earth iron garnet particles and bismuth-substituted rare earth iron garnet particles.  [7] The magnetic composite particles according to claim 5, wherein the particles having magnetism are particles obtained by applying a ferromagnetic substance to ferromagnetic oxide particles.  [8] The magnetic composite particles according to claim 7, wherein the magnetic composite particles are silica, an ionic polymer, and a Z or ionic surfactant.  [9] The magnetic composite particle according to claim 8, wherein the magnetic composite particle is a polymer having a carboxylic acid group, a sulfonic acid group, and a Z or phosphate group.  [10] The magnetic composite particle according to claim 8, wherein the ionic surfactant is a surfactant having a carboxylic acid group, a sulfonic acid group and a Z or phosphate group.  [II] The magnetic composite particle according to any one of claims 110, wherein the weight ratio of the magnetic particle to the chitosan layer is 100: 5-100: 300.  12. The weight ratio of the magnetic particles to the chitosan layer is from 100: 10 to 100: 90. 10. The magnetic composite particles according to any one of items 10 to 10. [13] The magnetic composite particle according to any one of [1] to [12], wherein the thickness of the thinnest portion of the chitosan layer is 5 nm or more. 14. The magnetic composite particles according to claim 11, wherein the specific surface area of the magnetic composite particles is in the range of 100 m ² / g. [15] The magnetic composite particle according to any one of claims 114, wherein the coercive force of the magnetic composite particle is in the range of 0.80 to 15.92 kAZm (10 to 200 Oersted). [16] The saturation magnetization of the magnetic composite particles is 5—80 A'm ² / 1¾

The magnetic composite particle according to any one of claims 1 to 15, wherein the magnetic composite particle has a range of: [17] a) a step of dissolving chitosan in water using an acid, b) a step of dispersing magnetic particles in the obtained chitosan solution, and c) a step of dispersing the obtained dispersion with a weak alkali base. And gradually neutralizing the particles. [18] The method for producing magnetic composite particles according to claim 17, further comprising: d) a step of adding a chitosan crosslinking agent into the system and stirring the mixture. [19] The method for producing magnetic composite particles according to claim 17 or 18, further comprising the step of: e) immediately neutralizing the neutral dispersion after step c with a weak alkaline base. [20] The method for producing magnetic composite particles according to any one of claims 17 to 19, wherein a protective colloid is added into the system before step c.