WO2005087367A1 - Particule composite magnétique et son procédé de production - Google Patents

Particule composite magnétique et son procédé de production Download PDF

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WO2005087367A1
WO2005087367A1 PCT/JP2005/004441 JP2005004441W WO2005087367A1 WO 2005087367 A1 WO2005087367 A1 WO 2005087367A1 JP 2005004441 W JP2005004441 W JP 2005004441W WO 2005087367 A1 WO2005087367 A1 WO 2005087367A1
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particles
chitosan
magnetic
magnetic composite
composite particles
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PCT/JP2005/004441
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Japanese (ja)
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WO2005087367A8 (fr
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Kenji Kohno
Mikio Kishimoto
Yoshiaki Nishiya
Masahiro Kusumoto
Shin-Ichiro Nishimura
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Hitachi Maxell, Ltd.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier

Definitions

  • 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.
  • Chitosan has conventionally been used for various purposes such as separation and purification because it has the property of adsorbing various substances.
  • chitosan has an amino group, its application to various fields has been attempted by immobilizing various functional groups and substances.
  • 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.
  • one drop is basically one composite particle, a large composite particle cannot be obtained with respect to the magnetic fine particles.
  • 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.
  • a structure in which a single composite particle includes a plurality of magnetic particles causes a thick portion and a thin portion of chitosan.
  • the internal magnetic substance may be partially exposed.
  • 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.
  • this example reduced the chitosan ratio and did not assume that a thick coating would be formed.
  • 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.
  • 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).
  • 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.
  • 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)
  • 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! ⁇ To provide composite materials! Puru.
  • 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.
  • a weak alkali base as a reagent for chitosan precipitation
  • 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.
  • a desirable mode of the present invention also has the following constituent powers.
  • 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-.
  • 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.
  • 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 above item 5, wherein the particles having magnetism are particles obtained by coating ferromagnetic iron oxide particles with an aionic substance.
  • 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.
  • aionic surfactant is a surfactant having a carboxylic acid group, a sulfonic acid group and a Z or phosphate group.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the properties as chitosan for example, the property of adsorbing various substances and fixing them are exhibited.
  • 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.
  • 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.
  • chitosan has a feature that the thickness of the magnetic material is small and the internal magnetic material is hardly exposed.
  • 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.
  • 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.
  • 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.
  • a chitosan solution obtained by dissolving chitosan by making it acidic may be gradually added by a method such as a dropping method.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • chitosan is generally insoluble in neutral or alkaline water
  • the covered chitosan can be used in a wide range without cross-linking.
  • 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.
  • 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.
  • 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.
  • 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.
  • a catalyst if necessary, a reactive group activator, a buffer, etc. may be used. May be.
  • a tertiary amine is used for the reaction between the amino group and the epoxy group
  • a dehydrating agent or carbodiimide is preferably used for the reaction between the amino group and the carboxyl group.
  • the crosslinking reaction may be performed by heating to a temperature required for the reaction.
  • 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.
  • 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.
  • 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.
  • a concentrated alkaline solution such as 45% NaOH
  • 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.
  • 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.
  • 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.
  • 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.
  • 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! ⁇ 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 ( ⁇ -Fe 2 O 3
  • At least one selected from the group consisting of one net particle and bismuth-substituted rare earth iron garnet particles is preferred.
  • magnetite particles are optimal because they have good magnetic field sensitivity when collected by a magnet with a large saturation magnetization.
  • chitosan is a cationic substance
  • 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.
  • particles having a strong ionization property such as mag hematite can easily obtain a uniform coating without any particular surface treatment.
  • 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.
  • 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.
  • 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.
  • the ratio of chitosan is lower than 100: 5, it becomes difficult to cover the whole of the magnetic particles.
  • 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.
  • 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.
  • the chitosan layer 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
  • 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 ⁇ of a physiologically active substance is increased, it is preferably used.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the method for producing ferromagnetic iron oxide particles used as the particles having magnetism is not particularly limited.
  • 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.
  • a NaOH aqueous solution was added to a divalent aqueous Fe ion solution in which ferrous sulfate (FeSO6 ⁇ ) was dissolved.
  • 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.
  • 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 ( ⁇ -FeOOH) particles are likely to be generated.
  • 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.
  • 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.
  • a typical production method is as follows.
  • chitosan 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.
  • 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.
  • cross-linking of chitosan is performed.
  • 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.
  • a protective colloid may be added into the system before the reaction for precipitating chitosan.
  • 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.
  • the protective colloid include polyvinyl phenol renore, methinoreseno reloose, hydroxyethino reseno reloose, hydroxypino pinole reloose, and the like.
  • 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).
  • 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.
  • 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.
  • a chitosan coating layer having a uniform and sufficient thickness is provided on the surface of magnetic particles.
  • the surface area can be increased, and even if the ratio of particles having magnetic properties inside is increased, the exposure can be suppressed.
  • magnetic particles are dispersed in a chitosan solution dissolved using an acid, and gradually neutralized using a weak alkali base.
  • 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.
  • 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 ⁇ m, 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.
  • chitosan 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.
  • PSH-80 manufactured by Yaizu Suisan Chemical Co., Ltd., deacetylation degree of 80% or more
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • PAA polyacrylic acid
  • 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.
  • 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.
  • OA oleic acid
  • 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.
  • 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.
  • Magnetite having an average particle size of about 0.02 ⁇ m 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.
  • 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.
  • 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.
  • Example 1 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.
  • 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.
  • the specific surface area was measured using a specific surface area / pore distribution measuring device (“SA3100” manufactured by Coulter Inc.).
  • the ferrite magnet 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.
  • the time until the composite particles are collected by the magnet portion and the liquid becomes almost transparent is measured.
  • 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, ⁇ and ⁇ .
  • 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.
  • 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.
  • the magnetic composite particles of Example 114 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.
  • 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.
  • the magnetic composite particles of the present invention can be used for immobilizing a physiologically active substance.
  • diagnostic drug carriers 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.

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Abstract

Particules composites magnétiques d’une taille moyenne de 0.01 to 4 mm comprenant des particules magnétiques et, superimposées sur les surfaces, des couches de chitosane. On fournit en particulier des particules composites magnétiques de structure à virole de cœur comprenant des particules magnétiques comme cœurs et des couches de chitosane comme virole, dont les particules magnétiques sont capables d’immobiliser des substances physiologiquement actives à haut rendement et après l’immobilisation, montrent une haute collectibilité par magnétisme. En outre, on fournit un procédé de production de particule composite magnétique composée de particules magnétiques et, surimposées sur les surfaces, des couches de chitosane, dont le procédé comprend les étapes de (a) dissolution du chitosane dans l’eau par acidification ; (b) dispersion des particules magnétiques dans la solution de chitosane et (c) lente neutralisation de la dispersion avec une base alkali faible.
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CN101240075B (zh) * 2008-03-07 2011-04-20 哈尔滨工业大学 壳聚糖磁性微球的制备方法及磁性微球固定化酵母的方法
CN102863655A (zh) * 2012-09-24 2013-01-09 厦门大学 一种链球状磁性壳聚糖/醋酸纤维素及其制备方法
JP2014133677A (ja) * 2013-01-09 2014-07-24 Shimane Univ 水溶性超常磁性ナノ粒子
CN104874366A (zh) * 2015-05-29 2015-09-02 西北师范大学 壳聚糖磁性吸附材料的制备及在吸附污水中Pb2+、As3+的应用
JP2016527881A (ja) * 2013-07-08 2016-09-15 バイオライト・ワールドワイド・カンパニー・リミテッド タンパク質、ポリペプチド、またはオリゴペプチドの固定化のための複合担体、その調製方法、および適用
JP2017155006A (ja) * 2016-03-02 2017-09-07 株式会社コスモビューティー 複合粒子、化粧料組成物、及び、複合粒子の製造方法
CN114700058A (zh) * 2022-04-29 2022-07-05 新疆师范大学 多孔磁性复合吸附剂的制备方法及其吸附二甲基胂酸的应用

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CN106268556B (zh) * 2016-08-16 2021-06-08 康盈红莓(中山)生物科技有限公司 一种蛋白纯化用磁珠的制备方法

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CN101240075B (zh) * 2008-03-07 2011-04-20 哈尔滨工业大学 壳聚糖磁性微球的制备方法及磁性微球固定化酵母的方法
CN102863655A (zh) * 2012-09-24 2013-01-09 厦门大学 一种链球状磁性壳聚糖/醋酸纤维素及其制备方法
JP2014133677A (ja) * 2013-01-09 2014-07-24 Shimane Univ 水溶性超常磁性ナノ粒子
JP2016527881A (ja) * 2013-07-08 2016-09-15 バイオライト・ワールドワイド・カンパニー・リミテッド タンパク質、ポリペプチド、またはオリゴペプチドの固定化のための複合担体、その調製方法、および適用
CN104874366A (zh) * 2015-05-29 2015-09-02 西北师范大学 壳聚糖磁性吸附材料的制备及在吸附污水中Pb2+、As3+的应用
JP2017155006A (ja) * 2016-03-02 2017-09-07 株式会社コスモビューティー 複合粒子、化粧料組成物、及び、複合粒子の製造方法
CN114700058A (zh) * 2022-04-29 2022-07-05 新疆师范大学 多孔磁性复合吸附剂的制备方法及其吸附二甲基胂酸的应用
CN114700058B (zh) * 2022-04-29 2023-09-19 新疆师范大学 多孔磁性复合吸附剂的制备方法及其吸附二甲基胂酸的应用

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