JP2002131320A - Ferrite-immobilized organism substance and its making method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To make magnetically manipulatable organism substances by immobilizing ferrite to the organism substances while keeping the activity of the organism substances and to realize wide applications thereof. SOLUTION: Magnetism is given to organism substances by coveringly immobilizing ferrite on them by ferrite plating at a temperatures and pH for keeping active the organism substances including; physiologically active substances such as lipid, proteins, antigens, antibodies, enzymes, and hormones; receptors relative to chemical substances such as drugs; and drugs.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biological material provided with magnetism by fixing ferrite which is a magnetic material, and a method for producing the same.

[0002]

BACKGROUND OF THE INVENTION Many common biological materials are non-magnetic and cannot be manipulated magnetically. If a biological substance can be fixed to ferrite, which is a magnetic substance, it becomes possible to magnetically manipulate the biological substance. As a biological material capable of being magnetically operated, as schematically shown in FIG. 3, an island-shaped ferrite layer 32 is formed on the surface of a polymer microsphere 34 by ferrite plating, and the ferrite surface is formed on the surface of the ferrite. Examples include enzyme immunological reagents such as cancer diagnostic reagents prepared by immobilizing antibody 31 which is a biological substance (Abe: Metals, Vol. 68, p. 290). This enzyme immunoreagent has the characteristic that it can be magnetically separated by utilizing the magnetism of ferrite, so that it can be processed at high speed as a reagent and can obtain high resolution. Also, a magnetic transport anticancer agent having a similar structure is known. In this method, an anticancer drug is induced into cancer cells in an affected area by magnetic operation, and the cancer cells are killed.

In the production of the above enzyme immunoreagents and magnetic transport anticancer agents, first, ferrite is immobilized on the surface of polymer microspheres, and then a biological substance is immobilized on the ferrite surface in another step. Are performed in this order. This is because the temperature at which ferrite is generated and fixed is high or the pH is far away from neutral, etc.
This is because the ferrite cannot be coexisted without losing the activity of the biological material when ferrite is generated. For example, in the case of performing ferrite plating on a polymer sphere, the reaction is performed at a temperature of 60 to 100 ° C. This temperature is not suitable for maintaining the activity of ordinary biological materials. As a method for producing ferrite from an aqueous solution, a coprecipitation method in which an alkali is added to an aqueous solution containing metal ions constituting ferrite to generate ferrite is known, but in this case, alkali addition is performed. Since it is necessary to increase the pH, for example, to 12 or more,
Also in this case, it is not suitable for maintaining the activity of a normal biological substance.

[0004]

SUMMARY OF THE INVENTION The present inventors have focused on the fact that it is desirable to directly generate and fix ferrite on a biological material to impart magnetism without impairing the activity of the biological material. Did research. If ferrite can be directly generated and fixed on individual biological materials, it differs from the conventional magnetically operable biological materials of the type in which the biological materials are fixed to particles such as polymer spheres in the case of the enzyme immunoreagent. It is expected that a new type of magnetically operable biological material can be obtained, which will enable a wide range of applications.

In order to produce and fix ferrite on a biological material without impairing the activity of the biological material, ferrite is formed in an aqueous solution containing the biological material, and the temperature and pH of the aqueous solution for ferrite formation are adjusted to the biological material. It is necessary to do within the range. In addition, the activity of the biological material must not be lost by fixing the ferrite.

The present invention provides a biological material having ferrite immobilized thereon and having magnetism, and a method for producing the same. The present invention has been accomplished by solving each of the above problems. is there.

[0007]

The ferrite-immobilized biological material of the present invention is characterized in that ferrite is fixed by ferrite plating to a portion of a biological material having no biological activity that does not inhibit the biological activity, and has biological activity and magnetism. It is characterized by the following.

FIG. 1 schematically shows the ferrite-fixed biological material of the present invention. In FIG. 1A, a biological material 1 is fixed to a ferrite 2.

In the present invention, a biological substance having a physiological activity is a substance which exhibits activity in a living body and has an affinity or an interaction with another substance in the living body or outside the living body. Proteins, antigens, antibodies,
In addition to substances originally possessed by the living body, such as physiologically active substances such as enzymes and hormones, and receptors for chemical substances such as drugs and environmental hormones, they are introduced into the living body from outside, such as drugs and endocrine disrupting substances. This includes those having an activity with other substances in the body, and composite structures obtained by combining these biological substances to form a composite.

In the present invention, the site where the ferrite of the biological material is fixed is a site where the specific biological activity possessed by the biological material is not impaired by the fixing of the ferrite. A hydroxyl group or the like is present in this portion, and the ferrite can be fixed by ferrite plating.

In the ferrite-fixed biological material of the present invention, the fixation of the ferrite may be performed via the spacer 3 as shown in FIG. The spacer may have a structure in which one end has a binding property with a biological substance and the other end has a group such as a hydroxyl group and a group capable of ferrite plating. Also, by providing a spacer, a spatial distance is provided between the immobilized ferrite and the biological material, and the ferrite immobilized via the spacer on the biological material is adapted to the specific activity inherent in the biological material. It can also have no effect.

In the present invention, the composition of the ferrite immobilized on the biological material is not particularly limited. In addition to magnetite (Fe ₃ O ₄ ) having good compatibility with the living body, at least a part of the metal element of magnetite may be used, if necessary. Various ferrite compositions substituted with one other metal element can be applied.

The size of the ferrite immobilized on the biological material is not particularly limited. However, in order to reduce the influence on the activity of the biological material, the size of the ferrite is preferably not too large as compared with the size of the biological material. It is preferred that it is not too small compared to the size of the biological material to allow manipulation. For this reason, for example, 1/10 of biological material
The size is preferably up to 10 times, more preferably 1/5 to 5 times the size of the biological material.

The surface of the ferrite fixed to the biological material may be coated with a resin or the like as necessary. As the resin used here, for example, glycidyl methacrylate can be preferably used.

As the ferrite fixed to the biological material of the present invention, a ferrite having superparamagnetism can be preferably used. If the biological material is small, for example, about 1 to 5 nm, and if the size of the ferrite to be fixed thereto is selected to be small in accordance with this, the ferrite becomes superparamagnetic due to the influence of thermal disturbance, and exhibits residual magnetization and It loses coercive force. Even if it is superparamagnetic, it can be magnetically operated by applying a magnetic field, and if it is superparamagnetic, it has no residual magnetization or coercive force, so there is no risk of magnetic aggregation in the absence of a magnetic field .

The production of the ferrite-immobilized biological material of the present invention can be performed by subjecting the biological material to ferrite plating to generate and fix ferrite at a predetermined portion of the biological material.

The fixation of ferrite to biological material is performed by
A biological substance is immersed in an aqueous solution containing ferrous ions, and metal ions such as ferrous ions are adsorbed to predetermined portions of the biomaterial, and the ferrous ions are oxidized to form a spinel ferrite phase. Things. In the present invention,
As a ferrite plating condition, the pH of the reaction solution is 40 ° C or less by selecting the pH as a condition close to neutrality of 7 to 9.
Ferrite can be fixed at room temperature or lower, and even at a temperature of about 5 ° C.

As described above, as the temperature range of the reaction solution in the present invention, a temperature range in which metal ions for ferrite plating can move at 40 ° C. or less can be used.
The temperature range of the reaction solution is more preferably 40 ° C. or less and 0 ° C. or more, and still more preferably 25 ° C. or less and 0 ° C. or more. In addition, since the oxidation conditions at this time may be mild conditions such that oxygen in the air is taken in by stirring, without altering the biological material or impairing its activity,
Ferrite can be immobilized on biological material.

FIG. 2 is a view schematically showing one embodiment of the method for producing a ferrite-fixed biological material of the present invention. In FIG. 2, the ferrite is fixed to the biological material 1 by immersing the biological material 1 in an aqueous solution 5 containing ferrous ions in a container 4 so that metal ions, such as ferrous ions, The ferrous ions are oxidized to form a spinel ferrite phase. Here, the pH is controlled by using a temperature / pH controller 7 to adjust the pH by adding an alkali. The oxidation is performed by stirring the liquid with a stirrer and taking in air.

In the present invention, the ferrite plating can be carried out at a pH of 7 to 9 as a ferrite plating condition so as not to impair the activity of the biological material, and the temperature of the reaction solution is room temperature 2.
It can be performed at 5 ° C or less. For example, it can be performed at a low temperature of about 5 ° C. The reaction can also be performed by adjusting the osmotic pressure of the reaction solution to a value suitable for the biological substance.

In the present invention, ferrite is fixed to a biological substance by using ferrite plating. Therefore, first, divalent iron ions are first adsorbed and bound to an adsorption site of an iron ion in the biological substance, for example, a position having a hydroxyl group. Since (part of) this ferrous ion is oxidized to form ferrite having a spinel structure, the position where ferrite is generated and fixed can be specified, and the fixing is strong.

The ferrite-immobilized biological material of the present invention has activity as a living body and can be magnetically operated from the outside, so that it can be used in many applications.

Among these many uses, the use as a carrier or a bioprobe for selectively capturing a biospecific affinity substance is one of the important ones. Specific examples of such a biospecific affinity substance carrier include:
There is a biospecific affinity substance carrier used for identification and separation / extraction of a receptor (receptor) in a living body that specifically binds to a chemical substance such as a drug or an endocrine disrupting substance in the living body. This can be achieved by fixing the ferrite to the chemical.

A chemical substance given to a living body binds to a specific receptor in the living body and acts on the living body after its structure and its function are changed. Therefore, it is necessary to search for and identify that particular receptor and clarify the relationship between the chemical and that receptor. In addition, if such a relationship becomes clear and the signal transduction pathway in cells from external stimuli can be elucidated, for example, by targeting a specific signal transduction pathway, the development of new drugs with large efficacy and few side effects and the development of new drugs It can be useful for design.

When a ferrite-immobilized carrier, which is one embodiment of the ferrite-immobilized biological material of the present invention, is used, the carrier is mixed with a cell fluid mixture to form a receptor for the chemical present in the mixture. After binding to the carrier, the receptor for this chemical can be conveniently separated and extracted with the carrier using magnetic separation.

Affinity chromatography has conventionally been used to separate and extract biomolecules such as a receptor for a specific drug from receptors in cells, but it requires a lot of labor and time. Recently, a separation and extraction method described in JP-A-10-195099 has been developed and used as a new separation and extraction method for biomolecules.

This uses microspheres in which a physiologically active substance having specific affinity is bound as a ligand to latex particles of a styrene-glycidyl methacrylate polymer via a spacer as a carrier, so that efficient separation and extraction can be achieved. It is possible to do so, and significant speedup and simplification are realized.

However, in order to separate the microspheres from the mixture, a sedimentation method such as centrifugal sedimentation is used. In the sedimentation method, it is necessary to pay attention to contaminants that are sedimented at the same time.
On the other hand, if the ferrite-immobilized biological material of the present invention is used as a carrier for a biospecific affinity biological material, the carrier can be separated from the mixture by magnetic separation. And the contamination can be avoided by magnetic separation.

The biospecific affinity substance carrier using the ferrite-immobilized biological substance of the present invention can be magnetically manipulated to construct a library of biospecific affinity substances or for specific purposes. Can be used for screening for a biospecific affinity substance according to the above.

The ferrite-fixed biological substance of the present invention can be used as an enzyme immunoreagent as described above, or can be used for administration of a drug to an affected part. By fixing the ferrite to the drug, it is possible to operate it magnetically when administering the affected area to the affected area, so that if the drug is concentrated on the affected area, the drug can be used efficiently, and after treatment Since excessive ferrite-fixed substances and used substances can be recovered by magnetically operating them using a magnet or the like, side effects of the drug can be reduced.

In these applications, the substance such as a drug substance or a protein may be fixed to ferrite via a spacer, or the ferrite may be directly fixed to the drug substance. The above-mentioned uses of the ferrite-fixed biological material of the present invention are merely specific examples. Therefore, the use of the ferrite-fixed biological material of the present invention is not limited to these.

[0032]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Next, embodiments of the present invention will be specifically described based on examples. The following examples illustrate the embodiments of the present invention, and the present invention is not limited to the examples described here.

(Example 1) (Immobilization of ferrite on albumin) BSA (bovine serum albumin), which is a kind of albumin, was selected as a biological substance.
The ferrite was fixed by ferrite plating.

Here, ferrite plating was performed under the following conditions. Ferrous chloride (FeC)
l ₂ ) and an aqueous solution of ferric chloride (FeCl ₃ )
The temperature of the aqueous solution was kept at a constant temperature of 25 ° C., and the aqueous solution was stirred to oxidize by taking in oxygen in the air. At this time, the pH of the aqueous solution was kept at 7 by adding ammonia water using a pH controller. In this way, BSA on which ferrite was fixed was obtained by plating for 10 minutes.

A part of the BSA on which the ferrite was fixed was taken out and observed with an electron microscope. As a result, it was confirmed that the ferrite was fixed on albumin. Further, as a result of X-ray diffraction of BSA to which the ferrite was fixed, formation of ferrite (magnetite having a spinel structure) was confirmed. In addition, as a result of performing a magnetic measurement on the BSA to which the ferrite was fixed, it was confirmed that the coercive force and the remanent magnetization were almost zero, and that the BSA exhibited superparamagnetic behavior.

(Example 2) (Immobilization of ferrite on E3330 albumin into which hydroxyl group was introduced) As a biological substance, 3- [5- (2,3-dimethoxy-6-methyl-1,4-
Benzoquinonyl)]-2-propenoic acid (a drug that selectively inhibits the transcription factor NFkB involved in rheumatism and inflammatory reactions 33
OH-E3330 obtained by introducing a hydroxyl group into 30 (Fujisawa Pharmaceutical, hereinafter abbreviated as E3330) was synthesized, and the hydroxyl group was subjected to ferrite plating to obtain a ferrite-fixed OH-E3330. This ferrite-immobilized OH-E3330 was tested for suppression of the transcriptional activation ability of NFkB, and was confirmed to have immunosuppression like E3330.

Ferrite plating for E3330 was performed under the same conditions as in Example 1. Ferrous chloride (FeCl ₂ ) and ferric chloride (FeC) are used as a reaction solution in a solution containing OH-E3330.
injecting an aqueous solution of l _3), maintaining the temperature of the aqueous solution at a constant temperature of 25 ° C., was oxidized by incorporating oxygen in air followed by stirring of the aqueous solution. At this time, using a pH controller and adding ammonia water,
The pH was kept at 7. E3330 to which ferrite was fixed was obtained by such plating.

Next, a part of the E3330 to which the ferrite was fixed was taken out and observed with an electron microscope. As a result, it was found that the ferrite was fixed. Further, as a result of performing X-ray diffraction of E3330 to which the ferrite was fixed, formation of ferrite (magnetite having a spinel structure) was confirmed.
Further, as a result of magnetic measurement of the E3330 to which the ferrite was fixed, it was confirmed that both the coercive force and the remanent magnetization were almost zero, and that the ferrite exhibited superparamagnetic behavior.

Example 3 (E3 with Ferrite Fixed)
Separation and Extraction of Receptor Using 330) Next, E3330 in which ferrite prepared in Example 2 was fixed.
Was used to separate and extract the receptor. The details of the following procedure followed the method described in JP-A-10-195099.

The culture solution of the Junker cells in suspension culture was centrifuged to collect the cells, and washed to remove impurities. The cells were expanded, the cell membrane was disrupted, and separated into a cytoplasmic fraction and a nuclear fraction. Next, the nucleus of the nuclear fraction was loosened, the nuclear components were extracted, and dialysis was performed to obtain a nuclear fraction extract.

The ferrite-immobilized E3330 was mixed with the nuclear extract, allowed to stand, and allowed to bind to a substance that specifically binds to the E3330, and then subjected to magnetic separation using a neodymium magnet. E3330 to which ferrite was fixed was separated. The separated ferrite-immobilized E3330 was washed and magnetically separated twice to separate and extract the receptor of E3330, a biological substance bound to this E33330, and dissociated and eluted with a buffer solution containing KCl to extract the receptor. . This receptor was measured by electrophoresis and found to have a molecular weight of approximately 38 kDa.
The protein band was confirmed.

Thus, the ferrite-fixed E333
It was found that the nuclear redox protein Ref-1 could be extracted using 0. Ref-1 reduces NFkB in the nucleus to enhance and activate its DNA binding ability. E
3330 inhibits its reducing activity by selectively binding to Ref-1 in vivo.

(Example 4) (Immobilization of ferrite on DNA) CREP / A which is involved in life phenomena such as generation and long-term memory and is composed of a plurality of transcription factor groups recognizing the same base sequence
TF (cAMP responsible element binding protein / act
A DNA having a plurality of ivating transcription factor binding sequences in tandem and having a single strand at the end was prepared. A spacer having a hydroxyl group was connected to the end of the DNA, and the hydroxyl group of the spacer was subjected to the ferrite plating described in Examples 1 and 2 to fix the ferrite, thereby obtaining a DNA with the ferrite fixed. . As a result of magnetic measurement of the DNA in which the ferrite was immobilized, it was confirmed that the coercive force and the remanent magnetization were almost zero, and that the ferrite exhibited superparamagnetic behavior.

Example 5 (DNA Immobilized with Ferrite)
Separation and Extraction of Biological Substances Using DNA) The ferrite-immobilized DNA prepared in Example 4 and HeLa cell nucleus extract were mixed, and a binding reaction was performed under physiological conditions. Thereafter, by repeating the separation by magnetic separation and the washing using a buffer solution, the DNA in which the ferrite was immobilized and the biological material was bound could be easily separated and purified. Subsequently, the ferrite is fixed by increasing the salt concentration of the buffer solution.
Biological substances bound to DNA could be dissociated. The biological material obtained in this way was identified and confirmed to be CREB / ATP.

As a result, it was found that CREB / ATP can be separated and extracted by using a plurality of CREP / ATF binding sequences in tandem and using ferrite-immobilized DNA.

[0046]

According to the present invention, by using ferrite plating,
Ferrite can be formed and fixed on a biological material while maintaining the activity of the biological material. The biomaterial having the ferrite immobilized thereon can be used as a carrier capable of selectively capturing a biospecific affinity substance and magnetically separating it, thereby greatly simplifying the operation of separating and purifying the biomaterial. It can also be used for construction and screening of a library of biospecific affinity substances to simplify it.

Further, the ferrite-fixed biological material of the present invention can be used for administration of a drug to an affected area. Since the ferrite is fixed to the drug, the drug can be magnetically guided to the affected area, the drug can be concentrated on the affected area, and the diffusion of the drug to other areas can be prevented, thereby preventing the side effects of the drug. it can.

[Brief description of the drawings]

FIG. 1 is a view schematically showing one embodiment of a ferrite-fixed biological material of the present invention.

FIG. 2 is a diagram schematically illustrating a method for producing a ferrite-fixed biological material of the present invention.

FIG. 3 is a diagram schematically showing a conventional reagent in which ferrite is fixed to spherical particles, and a biological substance is fixed to the surface of the ferrite.

[Explanation of symbols]

1 ... biological material, 2 ... ferrite, 3 ... spacer, 4 ... container, 5 ... reaction liquid, 6 ... stirrer, 7 ... temperature / pH controller.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Kazuhiro Nishimura 2-12-1 Ookayama, Meguro-ku, Tokyo F-term within Tokyo Institute of Technology 4B029 AA07 AA21 AA23 BB15 BB16 BB17 BB20 CC03 FA15 4B033 NA22 NA42 NA43 NA45 NB02 NB12 NB22 NC04 NC12 4B063 QR82

Claims (4)

[Claims] 1. A ferrite-fixed biological material, wherein a ferrite is fixed to a portion of a biological material having physiological activity that does not inhibit the biological activity by ferrite plating, and has bioactivity and is magnetic. 2. A ferrite-fixed biological material, wherein the ferrite is fixed via a spacer. 3. The ferrite-immobilized biological material, wherein the ferrite has superparamagnetism. 4. A method for producing a ferrite-fixed biological material, comprising a step of performing ferrite plating on the biological material at a temperature of 40 ° C. or less and a pH of 7 to 9 to fix ferrite to a predetermined portion of the biological material. .