JPS59195161A - Magnetic particle and its production - Google Patents

Abstract

PURPOSE:To obtain magnetic particles suitable for immobilizing of a living body component by adjusting a soln. mixture contg. gelatin, water soluble polysaccharide, metaphosphate and pulverous ferromagnetic material particles in a specific pH range then forming the insoluble particles within a specific grain size range by an aldehyde crosslinking agent. CONSTITUTION:Pulverous ferromagnetic material particles are dispersed in an aq. soln. contg. sodium metaphosphate and an anionic surface active agent. The dispersion is added to the mixed soln. obtd. by adding an aq. soln. of polysoccharide such as gum arabic removed of insoluble matter to an aq. soln. of gelatin adjusted with pH 9 and further mixing an aq. soln. of C2H5OH. The gelatin soln. dispersed therein with the magnetic particle is adjusted to 2.5-6pH with an aq. acetic acid and after the soln. is cooled to about 5 deg.C, a crosslinking agent such as glutaraldehyde or the like is added under stirring thereby obtaining hydrophilic and insoluble particles having 1-100mu grain size are obtd. Such particles are utilized for a solid carrier, etc. which permit easy operation such as washing and separation by taking advantage of the magnetic characteristic when a living component such as antigen, antibody or the like is going to be immobilized.

Description

[Detailed Description of the Invention] The present invention relates to novel particles that contain ferromagnetic fine particles and are capable of immobilizing various proteins such as antigens, antibodies, and enzymes, complex proteins such as glycoproteins, and sugars. This relates to a manufacturing method.

Particles containing ferromagnetic particles inside are expected to have various applications in the medical and pharmaceutical fields. As a method for producing it, for example, there is a method by K+' Widder et al. in which particles are coated with serum albumin in a magnetite and then emulsification and heat treatment are repeated, but this method is complicated in operation; There was also a problem with the stability of the obtained particles. Therefore, currently research is mainly focused on methods using various styrene polymers or synthetic latexes such as polyacrylamide, but these methods also have the problem of being difficult to operate and difficult to obtain particles of uniform quality. The difficulty of this manufacturing method is that the ferromagnetic fine particles are extremely prone to agglomeration, and for this reason, it has been extremely difficult to manufacture cylindrical particles with a particularly high content of ferromagnetic fine particles.

The present inventors believe that the method for producing gelatin particles that we previously developed and applied for a patent for (Japanese Unexamined Patent Publication No. 57-153658, etc.) is extremely suitable as a method for producing these particles. The present invention was completed based on the discovery that homogeneous particles with a uniform content of .

That is, the present invention includes (1) gelatin, a water-soluble polysaccharide, metaphosphate ions, and ferromagnetic fine particles, the amine groups of the gelatin are crosslinked with aldehyde, and the particle size is 1 to 1.
100μ spherical, hydrophilic and insoluble particles, (
2) Add acid to a mixed solution containing gelatin, water-soluble polysaccharide, metaphosphate and ferromagnetic particles to pH 2.5~
The present invention relates to a method for producing particles, characterized in that the spherical particles formed in the solution are insolubilized by adjusting the particle diameter to 6.0 and then applying an aldehyde-based crosslinking agent to insolubilize the spherical particles formed in the solution.

Gelatin is a type of derived protein obtained from collagen. Among gelatins, acidic gelatin is particularly preferred.

Water-soluble polysaccharides can be used as thickeners or thickeners, and also include derivatives and salts of polysaccharides. Examples include gum arabic, carboxymethyl cellulose, sodium alginate, agar, carrageenan, etc., with gum arabic being particularly preferred.

Metaphosphate ions are anions formed when some cations of metaphosphate are separated, and the number of charges depends on the pH at which particles are formed, the type of metaphosphate used as a raw material, etc. It is determined. Metaphosphates include, for example, sodium mitsutaphosphate and sodium hexametaphosphate.

The ferromagnetic fine particles are fine particles of iron, cobalt, nickel, etc., and ferrite such as magnetite. It is well known that ferromagnetic fine particles with a diameter of approximately 50 to 500 mm are suspended almost uniformly in a solution by stirring, etc.
A particle size of about 80 is sufficient.

The ferromagnetic fine particles are preferably added after being dispersed in the liquid in advance, and anionic and nonionic surfactants and the like are used as appropriate to maintain the dispersion state. In such a dispersed state, there are Al and Tojitoferricolloid (manufactured by Kuiho Industries).

In the particles, water-soluble polysaccharides and metaphosphate ions are electrostatically bonded to gelatin, and the metaphosphate ions are cross-linked by aldehydes between the amine groups of gelatin. Although it is contained inside the particles, it is thought that it wraps around the surface in particular to form an electric double layer.Furthermore, the ferromagnetic fine particles are distributed throughout such particles.

Most of these particles are made of water, with a composition of about 2 to 10% gelatin, about 1 to 5% water-soluble polysaccharide,
Metaphosphate ions are about 0.01 to 5%, ferromagnetic fine particles are about 0.000001 to 10%, and water is about 60%.
It is about 90%. When the particles are colored, they contain a pigment, and depending on the manufacturing method, they may also contain surfactants, hydrophilic organic solvents, and the like.

The physical properties of the particles of the present invention are shown below.

(11 Electrophoretic mobility The electrophoretic mobility of the particles of the present invention in 0.15M phosphate buffered saline (hereinafter abbreviated as PBS) at pH 7.2 at 25°C is 0.7 to 1.5 μm. /sec
It's in /V/.

(2) External shape It is spherical with a diameter of 1 to 100μ.

A scanning electron micrograph at 3000x magnification is shown in Figure 1.

(3) Electron micrograph A 10,000x transmission electron micrograph is shown in Figure 2.

As shown in the figure, the ferromagnetic fine particles are distributed almost uniformly throughout the particles.

(4) Color: white to pale yellow and opaque unless specifically colored = (5) Attitude toward water: Hydrophilic (6) Immobilization of active protein The particles of the present invention are known to sensitize sheep red blood cells to antigens or antibodies. The straw can be sensitized by the method,
Other active proteins such as enzymes, lymphokines, etc. can be immobilized. Examples of such sensitization methods include phosphoric acid, formalin, glumeraldehyde, pyruvic aldehyde, bis-diazotized benzidine, toluene-2,4-
A method using diisocyanate and the like can be mentioned.

Furthermore, enzymes or various active proteins such as antigens and antibodies can be immobilized on the uninvented particles by a known method of immobilizing enzymes using amino groups, carboxyl groups, hydroxyl groups, etc. Examples of such immobilization methods include the soazo coupling method and the acid azidation method.

(7) Immobilization of complex proteins such as glycoproteins and sugars Complex proteins can be immobilized by the same method as in (6) for immobilization of active proteins. Furthermore, sugars can be fixed using, for example, an oxidizing agent such as periodic acid.

Such particles of the present invention can be obtained by adding an acid to a mixed solution containing gelatin, a water-soluble polysaccharide, a metaphosphate, and ferromagnetic particles while stirring to adjust the pH to 2.5 to 6.0. , and then insolubilized by the action of an aldehyde crosslinking agent.

In this case, the concentration of each substance in the solution before pH adjustment is about 0.01 to 5% for gelatin, preferably about 0.05 to 1.0%, and about 0.01 to 5% for water-soluble polysaccharide. , preferably 0.05 to 1.0 multiphase degree, and ferromagnetic fine particles of about 0.000001 to 15 inches.

The metaphosphate is preferably contained in an amount of about 1 to 3% of the dry weight of gelatin. The concentration of each substance may be appropriately determined within the range of concentration according to the particle size and physical properties of the desired particles.

In addition, a hydrophilic organic solvent, a surfactant, a coloring agent, etc. are appropriately added to the solution before pH adjustment.

Hydrophilic organic solvents can improve the physical properties of the particles produced, particularly their dispersibility and hardness. Examples of the hydrophilic organic solvent that can be used include lower alcohols, ethyl alcohol, ethyl alcohol, flobyl alcohol, and acetone, and the concentration is preferably about 4 to 25 volumes.

The surfactant is used to prevent agglomeration of the generated particles and maintain dispersion, and is preferably an anionic or nonionic surfactant.

Examples of anionic surfactants include alkyl sulfosuccinic acids, alkyl sulfomaleic acids, alkyl sulfates, and polyoxyethylene alkyl ether sulfates, and examples of nonionic surfactants include polyoxyethylene fatty acids. Ester, polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, 101J ethylene glycol JJit
Fats include esters. In the case of anionic surfactants, the concentration in the solution before pH adjustment is 0.001 to 0. In the case of a nonionic surfactant of about 1% O, an aggregation prevention effect can be obtained at a polyphasic degree of 0.01 to 0.1. By cooling the solution, aggregation can be prevented at lower concentrations.

If the particles are to be colored, a color promoter may be added to the solution before forming the particles. 2.1: An example of the case where the product of the present invention is used as a carrier for an indirect passive aggregation reaction. That is, since non-inventive products are usually white to pale yellow and opaque, it is possible to make the determination of the agglomerated image easier by coloring the stiffness. As a dark coloring agent, tr: A dye or the like can be used. However, reactive dyes such as reactive red and reactive blue are particularly suitable because the color does not fade when colored with reactive dyes. When adding a colorant, it is usually 0.005 to 0.
．． The amount is about 5%, but if a reactive dye is used, it will be reduced to about 1 to 555% of the dry weight of gelatin.

The process of preparing such a solution is not in question;
For example, each of the components except the ferromagnetic fine particles may be dissolved in muddy water and then mixed, or each component may be dissolved together. However, in order to facilitate the dissolution of each substance, hydrophilic solvents are often added afterwards; however, since fresh water-soluble polysaccharides often contain small amounts of insoluble components, they must be dissolved separately. Should I give it to you? Gelatin reacts with water-soluble polysaccharides at a pH above its isoelectric point, producing cloudiness, so when using powdered gelatin, add alkali to raise the pH of the solution to at least around that point. It is better. However, the table shows that even after this cloudiness occurs, it can be eliminated by adding alkali. In any case, the solution must be free of suspended matter other than the ferromagnetic particles before addition of the acid.

The ferromagnetic fine particles may be suspended by adding a predetermined amount at any stage of preparing such a solution.

The temperature of the solution must be above the gelatin temperature WJJ. This gluing temperature varies depending on the concentration of gelatin, but is usually about 25 to 30°C.

From the viewpoint of good particle formation, the temperature is particularly good at 35-50°C.Next, add acid to this solution while stirring to adjust the pH to 2.5-50°C.
Adjust to 6.0. This step is where particles are generated. 35-50℃ to form uniform particles
It is best to heat the mixture and add the acid dropwise while stirring moderately. Optimal p in the pH range of 2.5 to 6.0
) Since I varies depending on the composition of the raw material solution and the intended particle size, it is best to determine it by conducting experiments in advance. For example, when using the obtained IL particles as an antigen-sensitizing carrier,
The particle size should be about 10 μm, and the pH range is between 4.0 and 5.5 (Dgl).

This pHg, (the acid used for 1 adjustment is limited to 4--
The acid may be a non-rotating acid or a voiced acid, and preferably one that is as mild as possible, such as acetic acid.

The particles produced in this step do not disappear even if the temperature of the system is lowered below the gelatin gluing temperature, so there is no equilibrium relationship with the mother liquor.

After adding the acid, it is preferable to quickly cool the particle dispersion to prevent the formed particles from agglomerating. And the liquid temperature is 10
When the temperature drops below 0.degree. C., an aldehyde crosslinking agent is added to insolubilize the particles. The amount of this crosslinking agent added is about 0.1 to 200 g of the dry weight of gelatin, and after addition, the mixture is left to stand for about a minute to allow the crosslinking reaction to occur sufficiently. Examples of the crosslinking agent include (ri), glutaraldehyde, formaldehyde, glyoxal, crotonaldehyde, acrolein, and acetaldehyde, with glutaraldehyde being particularly preferred.

Treatment with aldehyde crosslinking agent y1. Afterwards, the particles are collected by centrifugation or by using magnetic force, and washed by 8 washes. The cleaning solution can be washed two or three times with water containing the same surfactant at the same concentration as the surfactant used for particle dispersion. However, if crosslinking is insufficient, it may swell in a salt solution. Therefore, when used for such purposes, it is recommended to treat it with an aldehyde-based crosslinking agent to prevent swelling. For example, when total antigen sensitization is required, nermarin treatment is performed under the conditions of fixation of red blood cells in a mildly saturated monophosphate solution.This treatment prevents swelling and uses the bactericidal effect of formalin. Particles that can be stored for a long period of time are obtained.

The unexploded particles should be uniformly dispersed in ferromagnetic fine particles or washed with IF81 to remove the magnetism.
Revenue? 9 It is easy to do. From the point where the trench is chemically and physically homogeneous and stable and has no antigenic activity, the carrier for the passive coagulation reaction is taken, - (preferably,
In this case, there is an advantage that the aggregation reaction time is shortened by about half. The particles of the invention are suitable as carriers for immobilizing proteins, complex proteins, sugars, etc., and by utilizing their magnetism, they can be expected to be used in various new applications. Such particles can be easily and inexpensively produced in large pieces with any particle size and content of several ferromagnetic particles within the above range.

Examples are shown below. In addition, in Unknown a Lu, the person in charge represents the person in charge of weight unless there is a special @e listing.

Example Acidic gelatin 41 having an isoelectric point of pH 9 was dissolved in warm water at 40° C. to a pH of 100° C., and the pH was adjusted to 9 using a 10% sodium hydroxide solution. gum arabic 4t
was dissolved in water to a volume of 100 ml, and after removing insoluble matter in a furnace, it was heated to 40°C.

Mix 25ml of the gelatin solution obtained in this way and 25ml of gum arabic solution, and add 4ml of this mixed solution in advance.
30 volume i% ethyl alcohol solution heated to 0°C 15
0- and stirred well. To this, solution 1-1 of sodium hexametaphosphate solution o, sy, to% alkyl sulfomaleic acid (trade name Demol Ep, manufactured by Kao Soap ■) and ferricolloid W- with an average particle size of 150A'' were added.
35 (product name, manufactured by Kuihoe Gyo@) 60μt (F
e 5.2■) or 300 μt (Fe 30
．． 4-++V-containing) was added and stirred well.

Next, while maintaining the temperature at 40° C., a 10 volume-3M% acetic acid solution was added dropwise to adjust the pH as shown in Table 1, thereby producing particles.

The particle dispersion obtained by this pH adjustment was cooled on ice and
℃, add 0.651 gel taraldehyde,
After stirring thoroughly, the mixture was allowed to stand overnight at this temperature.

The particle dispersion was then centrifuged at 2000 rpm for 10 minutes to collect particles as pellets.

A washing operation in which the particles were suspended in a 0.005% Demol Ep solution and centrifuged was repeated three times, then dispersed in 4 volumes of thiformin solution and left at 5° C. for one week.

The results obtained are shown in Table 1.

The sedimentation velocity of the thus-travelled particles was determined by changing the magnetic field, and the results are shown in Table 2. In addition, place the blood sedimentation tube 'fI: on a Qlj constant (-i magnet, and add any of the particles shown in the second column to the pf (0°15M1.l of 7,2) so that the concentration is 5 v/v%. The particle surface was uniformly suspended in acid-buffered saline and the particles had a particle size of 2.5 cm, 5.0 cm, and IQ.
Each time required for the o+ to settle was measured.

Table 2 Note that the true north value determined by the liquid phase displacement method is Fe, 0. ．． 0
In the case of particles containing ゜2 coefficient, it is 1.30 to 1.40,
1.50 for particles containing 1.2% Fe, Os
~1.80, Fe, 0. , in the case of particles not containing , it was 1.01 to 125.

[Brief explanation of drawings]

FIG. 1 is a scanning electron b0 micrograph of the uninvented particle, and FIG. 2 is a transmission 2Ti! ! This is an electron micrograph. Judgment - Notes Applicant Fujirebio Co., Ltd. Agent
Patent Attorney Masahiro Tanaka No. 1 Illustration Proceeding Amendment Office (Voluntary) September 1981 G Case 1 Display of Case 1989 Patent Application No. 69112 2 Name of Invention Magnetic Particles and Process for Producing the Same 3 Case of Person Who Amends Relationship with Patent applicant name Fujirebio Co., Ltd. 4 agent residence Address: 2-1 Kyobashi, Chuo-ku, Tokyo 104: 3rd edition Contents of 6th amendment (1) Specification, page 4, lines 6 to 8 "Ferromagnetic particles (fine particles such as ferrite, etc.)" is corrected as follows.U Ferromagnetic material refers to a substance that exhibits magnetic hysteresis.Specifically, iron, cobalt, ferrite, etc. Examples include metal magnetic materials such as Kel or their alloys, and oxidized metal magnetic materials such as ferrite (e.g. magnetite).'' Page 5, line 16 ``07~1.5J r, -o7~-1
5” page 5 line 19 “μ” “μm” page 16 table
"(μ)""(μm)J

Claims (1)

[Claims] 1. A gelatin containing gelatin, a water-soluble polysaccharide, a metaphosphate ion, and ferromagnetic fine particles, in which the amine groups of the gelatin are cross-linked with aldehyde, and the particles are spherical and hydrophilic with a particle size of 1 to 100μ. and insoluble particles 2 Add acid to a mixed solution containing gelatin, water-soluble polysaccharide, metaphosphate, and ferromagnetic fine particles to pH 2.5 ~
6] A method for producing particles, which comprises adjusting the particle size to 0 and then applying an aldehyde crosslinking agent to insolubilize the spherical particles formed in the solution.