JP2010207322A - Magnetic particle, method and apparatus for manufacturing the same, and magnetic particle for tumor cytoclasis, cytoclasis method, cytoclasis device, and therapeutic appartatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide magnetic particles in a new shape which are effectively used for a variety of purposes, such as selectively destructing tumor cells or the like, a method for manufacturing the same, and an apparatus for manufacturing the same. <P>SOLUTION: A magnetic particle 1 includes a core part 1 and many hair-like projections 3 around the core part 2, where the ratio of the length L of the hair-like projection 3 to a particle diameter D including the hair-like projections 3 is between 5% and 30%. The magnetic particles 1 whose average particle diameter D including the hair-like projections 3 is within the range from 100 nm to 300 nm are excellently obtained as iron particles formed by a gas flow sputtering method, and are used as magnetic particles for destructing tumor cells by a converted magnetic field given inside the tumor cells from outside by phagocytosis or endocytosis. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a magnetic fine particle having a novel shape, a manufacturing method thereof, and a manufacturing apparatus thereof. Furthermore, the present invention relates to a magnetic fine particle that can be preferably used to selectively destroy tumor cells, a cell destruction method, a cell destruction device, and a treatment device using the magnetic fine particle.

  Patent Document 1 below proposes a gas flow sputtering apparatus that can consume sputter vapor generated by sputtering as an ultrafine particle deposition film at a high rate of 50 atomic% or more. In this gas flow sputtering apparatus, it is described that the particle diameter and deposition rate of ultrafine particles of the order of several nanometers forming a deposited film can be controlled by adjusting the flow rate of the carrier gas.

  On the other hand, in Patent Document 2 below, particles that can be specifically bound to a target cell and can be moved when subjected to an external stimulus, and particles that are specifically bound to a target cell are given an external stimulus and the particles are attached. A cell disruption device having a target cell disruption means for moving and disrupting a target cell has been proposed. Furthermore, a particle-binding step that specifically binds to target cells by adding particles that can specifically bind to target cells and that can move when subjected to external stimuli into a sample containing target cells, and specific to target cells There has also been proposed a cell disruption method including a target cell disruption step in which externally stimulated particles are imparted with external stimuli to move particles to disrupt target cells.

JP 2000-87226 A JP 2004-49105 A

  (First Problem) The gas flow sputtering apparatus and method described in Patent Document 1 is a technique for generating fine particles of several nm and forming a deposited film with the fine particles. The present inventor succeeded in producing fine particles having a new shape which has not been conventionally obtained in the course of earnest research using the gas flow sputtering apparatus and method.

  A first object of the present invention is to provide a magnetic particle having a novel shape that can be effectively used for various applications, a method for producing the same, and a production apparatus therefor.

  (Second problem) In the cell disruption technique (apparatus and method) described in Patent Document 2, magnetic beads having a particle size of 0.5 to 10 μm are specifically bound to the surface of the target cell and bound. A target cell is destroyed by moving magnetic beads or the like in a magnetic field. However, the present inventor has found that tumor cells can be destroyed on a principle completely different from that of the document 2 by using magnetic fine particles having a new characteristic form.

  A second object of the present invention is to provide a magnetic particle having a novel shape that can be preferably used to selectively destroy tumor cells, a cell destruction method, a cell destruction device, and a treatment device using the magnetic particle. It is in.

  (1) A magnetic fine particle according to the present invention for solving the above-mentioned problems is composed of a core part and a number of beard-like protrusions around the core part, and the beard-like protrusion corresponding to the particle diameter including the beard-like protrusions. The length ratio is 5% or more and 30% or less.

  According to the newly completed magnetic fine particle of the present invention, it consists of a core part and a number of beard-like protrusions around the core part. Or, when a converted magnetic field is applied from the outside after endocytosis, it can cause physical vibration to selectively destroy only tumor cells, for example, to an antigen that is specifically expressed in the structure of living cells Affinity substances can be adsorbed or added to beard-like protrusions and transported to the relevant location, for example, using beard-like protrusions as magnetic separation particles for drug development, for example, using beard-like protrusions It can be used as a drug delivery.

  A preferred embodiment of the magnetic fine particles according to the present invention is configured such that the average particle diameter including the beard-like protrusions is in the range of 100 nm to 300 nm. Such extremely fine microparticles are, for example, phagocytosed or endocytosed by cells and easily taken up into cells.

  A preferred embodiment of the magnetic fine particle according to the present invention is configured such that the length of the whisker-like protrusion around the core portion is divided into a long region and a short region.

A preferred embodiment of the magnetic fine particles according to the present invention is configured to be iron fine particles formed by gas flow sputtering. These iron fine particles are fine particles having a high saturation magnetization of about 1490 emu / cm ³ and act sensitively by an external magnetic field.

(2) In the method for producing magnetic fine particles according to the present invention for solving the above-described problem, the sputtering vapor released from the target is transferred as a flow of a sputtering gas or a gas obtained by adding a carrier gas to the sputtering gas as required. Then, in the transfer process, the sputter vapor is condensed, and the magnetic fine particles obtained are recovered in a recovery part,
It has a transfer time adjusting means for extending the required time T of the transfer process until a large number of whisker-like protrusions are generated around the magnetic fine particles. According to this invention, magnetic fine particles having a large number of whisker-like protrusions around can be easily produced by the transfer time adjusting means.

  In the above invention, the transfer time adjusting means is a means for increasing the sputtering pressure to generate a turbulent flow in the gas flow, extending the transfer time from the target to the recovery part, and forming a mustache-like protrusion, or from the target It is preferable to configure so as to form a mustache-like projection by changing the distance to the collection unit.

  (3) A magnetic fine particle manufacturing apparatus according to the present invention for solving the above-described problems includes a hollow target that generates sputter vapor, a sputtering gas introduction hole that introduces a sputtering gas into the hollow target, The generated sputter vapor is transferred as a sputter gas introduced from the sputter gas introduction hole or a gas flow obtained by adding a carrier gas to the sputter gas as necessary, and the sputter vapor is condensed in the transfer process to obtain a magnetic An adjustment device that adjusts the transfer time until a large number of whisker-like protrusions are generated around the fine particles, and a recovery unit that recovers the magnetic fine particles are provided. According to the present invention, magnetic fine particles having a large number of whisker-like protrusions can be easily manufactured by the transfer time adjusting device.

  In the above invention, the adjusting device may increase the sputtering pressure to generate a turbulent gas flow, and increase the transfer time from the target to the recovery unit to form the mustache-like protrusions, or It is preferable that the transfer length variable device is configured to change the distance from the target to the recovery unit to form the mustache-like projections.

  (4) The magnetic fine particles for tumor cell destruction according to the present invention for solving the above-mentioned problems are composed of a core part and a large number of whiskers around the core part, and phagocytosis or endocytosis in the tumor cell. The tumor cells are destroyed by a conversion magnetic field applied from the outside.

  According to this invention, magnetic fine particles phagocytosed or endocytosed in tumor cells are different from spherical particles without beard-like projections, and are selected by causing a physical vibrational motion when an external conversion magnetic field is applied. It is possible to destroy tumor cells.

  In a preferred embodiment of the magnetic fine particle for tumor cell destruction according to the present invention, the ratio of the length of the whisker-like protrusion to the particle diameter containing the whisker-like protrusion is 5% or more and 30% or less. The average diameter is in the range of 100 nm to 300 nm.

  In a preferred embodiment of the magnetic fine particles for tumor cell destruction according to the present invention, the whisker-like process has a substance having affinity for or damage to a constituent protein or sugar chain antigen specifically expressed in a living cell structure. Is added. In the present invention, the substance having affinity for the constituent protein or sugar chain antigen is: (i) an antibody or affinity protein for the protein or sugar chain antigen inside or outside the cell membrane, or (ii) a ribosomal membrane antigen or lysosomal membrane. Antibody or affinity protein for antigen, (iii) BAX or P53 protein for mitochondrial membrane or protoplasm, and (iv) antibody or affinity protein for antigen distributed in nuclear membrane.

  According to this invention, a substance having an affinity or a hindrance to a constituent protein or a sugar chain antigen specifically expressed in the structure of a living cell is added to the beard-like projections (including the meaning of adsorption). Therefore, the magnetic microparticles phagocytosed or endocytosed in the cell each have a specific behavior, and a desired portion (cell membrane, lysosome, mitochondria, nuclear membrane, etc.) can be destroyed.

  (5) In the cell disruption method according to the present invention for solving the above-described problem, magnetic microparticles comprising a core portion and a number of bearded projections around the core portion are phagocytosed or endocytosed in the cell. And a step of applying a conversion magnetic field from the outside to apply physical stress to the cells to selectively destroy target cells to be destroyed among the cells.

  According to the present invention, magnetic particles phagocytosed or endocytosed in tumor cells are different from spherical particles without beard-like projections by applying a conversion magnetic field from the outside and applying physical stress to the magnetic particles. It is possible to selectively destroy tumor cells by causing a physical vibration movement.

  A preferred embodiment of the cell disruption method according to the present invention is configured such that the average particle diameter including the beard-like projections is in the range of 100 nm to 300 nm.

  (6) A cell disruption device according to the present invention for solving the above-mentioned problems is composed of a core part and a number of bearded projections around the core part, and is phagocytosed or endocytosed in the cell, and converted magnetic field And applying a physical stress to the magnetic fine particles that have been phagocytosed or endocytosed in the cells by a conversion magnetic field, and target to be destroyed among the cells. And a conversion magnetic field device that selectively destroys cells.

  According to the present invention, magnetic particles phagocytosed or endocytosed in tumor cells are different from spherical particles without beard-like projections by applying a conversion magnetic field from the outside and applying physical stress to the magnetic particles. It is possible to selectively destroy tumor cells by causing a physical vibration movement.

  In a preferred embodiment of the cell disruption apparatus according to the present invention, the ratio of the length of the bearded protrusions to the particle diameter including the bearded protrusions is 5% or more and 30% or less, and the average particle diameter including the bearded protrusions In the range of 100 nm to 300 nm.

  (7) A therapeutic apparatus according to the present invention for solving the above-described problems has at least the cell destruction apparatus according to the present invention, and controls applied magnetic field conditions of a conversion magnetic field apparatus provided in the cell destruction apparatus to control tumor cells. Is selectively destroyed.

  (1) According to the magnetic fine particle of the present invention, it consists of a core portion and a number of beard-like projections around the core portion. Therefore, unlike a spherical particle having no beard-like projections, When a converted magnetic field is applied from the outside after endocytosis, it can cause physical vibration to selectively destroy only tumor cells, for example, it has affinity for antigens that are specifically expressed in the structure of living cells Can be adsorbed or added to beard-like protrusions and transported to the corresponding location, for example, using beard-like protrusions as magnetic separation particles for drug development, or using, for example, beard-like protrusions It can be used as a delivery.

  (2) According to the method for producing magnetic fine particles of the present invention, magnetic fine particles having a large number of whisker-like projections can be easily produced by the transfer time adjusting means.

  (3) According to the apparatus for producing magnetic fine particles of the present invention, magnetic fine particles having a number of whisker-like protrusions around can be easily produced by the transfer time adjusting device.

  (4) According to the magnetic fine particles for tumor cell destruction of the present invention, the magnetic fine particles phagocytosed or endocytosed in the tumor cells are different from the spherical particles having no whisker-like projections when a conversion magnetic field is applied from the outside. It is possible to selectively destroy tumor cells by causing a physical vibration motion.

  (5) According to the cell disruption method of the present invention, the magnetic fine particles phagocytosed or endocytosed in the tumor cells are different from the spherical particles having no beard-like projections, and applied to the magnetic fine particles by applying a conversion magnetic field from the outside. By applying a physical stress, it is possible to cause physical vibrational motion and selectively destroy tumor cells.

  (6) According to the cell disruption device of the present invention, the magnetic fine particles phagocytosed or endocytosed in the tumor cells are different from the spherical particles having no beard-like projections, and are converted into magnetic fine particles by applying a conversion magnetic field from the outside. By applying a physical stress, it is possible to cause physical vibrational motion and selectively destroy tumor cells.

  (7) According to the treatment apparatus of the present invention, tumor cells can be selectively destroyed only by controlling the applied magnetic field conditions of the conversion magnetic field apparatus included in the cell destruction apparatus.

It is a schematic diagram which shows an example of the magnetic fine particle of this invention. (A) is an external view and (B) is a cross-sectional view. It is a transmission electron micrograph which shows the example of the magnetic fine particle of this invention. It is a transmission electron micrograph which shows the other example of the magnetic fine particle of this invention. 6 is a transmission electron micrograph showing still another example of the magnetic fine particles of the present invention. 1 is a schematic configuration principle diagram showing an apparatus for producing magnetic fine particles of the present invention. It is a schematic cross section which shows an example of the target holder periphery structure of the manufacturing apparatus of magnetic fine particles. In magnetic fine particles, it is explanatory drawing of the aspect divided into the area | region A and the short area | region B where the length of the mustache-like protrusion around a core part is long. It is typical explanatory drawing of the magnetic fine particle for tumor cell destruction of this invention. It is explanatory drawing of the magnetic fine particle for tumor cell destruction which added the antibody. It is typical explanatory drawing which shows the aspect which the magnetic particle (magnetic bead with a particle size of 4500 nm) for the tumor cell destruction of the past (Comparative Experimental example 5) adhered to the cell membrane. It is a result of the particle size distribution of the obtained magnetic fine particles. It is a result of the antitumor effect with respect to each tumor cell when using 4 types of magnetic particles in the comparative experiment example 6. FIG.

  Next, an embodiment of the present invention will be described. In addition, this invention includes the range including the technical idea, and is not limited to description, drawing, etc. which are shown below.

[Magnetic fine particles, method for producing the same, and apparatus for producing the same]
First, a magnetic particle having a novel shape that can be effectively used for various applications, a manufacturing method thereof, and a manufacturing apparatus thereof will be described.

(Magnetic fine particles)
As shown in FIGS. 1 to 4, the magnetic fine particle 1 is a fine particle composed of a core portion 2 and a number of mustache-like protrusions 3 around the core portion 2. It can be called fine particles. FIG. 1A is a schematic external view, and FIG. 1B is a schematic cross-sectional view. 2 to 4 are transmission electron microscope (TEM) images. The shape of the magnetic fine particle 1 is a true spherical shape, a spherical shape (excluding a true spherical shape), an elliptical spherical shape, or the like.

  The ratio of the lengths of the whisker-like protrusions 3 constituting the magnetic fine particles 1 is preferably 5% or more and 30% or less with respect to the average particle diameter D of the magnetic fine particles 1. The ratio (%) of this length is represented by [(average length L of whiskers 3) / (average diameter D of magnetic fine particles 1)] × 100. Here, the diameter D of the magnetic fine particles 1 can be measured from a transmission electron micrograph, and the average diameter is obtained by using the average value of the diameters D of the magnetic fine particles 1 measured from a plurality of directions (for example, four directions or six directions). It was. Further, the length L of the beard-like projections 3 can also be measured from a transmission electron micrograph, and the average length thereof is the length L of the plurality of beard-like projections 3 (for example, n = 5 or more, for example, about 5 to 20). Was measured and the average value was used.

  The magnetic fine particles 1 including the beard-like protrusions 3 have an average particle diameter D in the range of 100 nm to 300 nm. The magnetic fine particles 1 of the present invention can obtain extremely fine fine particles having an average particle diameter D in this range with good reproducibility. The magnetic fine particles 1 of the present invention can have the average particle diameter D in the entire range of 100 nm to 300 nm, and the range of 100 nm to 300 nm as the particle size distribution as shown in the experimental examples described later. The magnetic fine particles 1 having an average particle diameter of about 200 nm can be used. The average particle diameter D can be controlled according to the production conditions of the magnetic fine particles 1 described later. Details will be described later.

  When the average particle diameter D of the magnetic fine particles 1 is 100 nm or more and 300 nm or less, the diameter d of the core portion 2 is such that the ratio of the length L of the whisker-like projections 3 to the particle diameter D of the magnetic fine particles 1 is 5% or more and 30% or less. Therefore, the minimum is 40 nm (40% of 100 nm), and the maximum is 270 nm (90% of 300 nm). Under the conditions of the experimental examples described later, the diameter d of the core portion 2 was relatively large in the range from 140 nm to 180 nm (see Table 1 described later). This range can be arbitrarily adjusted according to the manufacturing conditions.

  The average length L of the mustache-like projections 3 is 5 nm (5% of 100 nm) since the ratio of the length L of the mustache-like projections 3 to the particle diameter D of the magnetic fine particles 1 is 5% or more and 30% or less. ) And a maximum of 90 nm (30% of 300 nm), but some outside this range may also be included. On the other hand, the range of the length L of the individual whisker-like protrusions 3 around the magnetic fine particles 1 is not the average, but is a minimum of about 2 nm and a maximum of about 150 nm. Many (see Table 1 below). Further, the diameter of the beard-like projection 3 is a very fine diameter of 5 nm to 15 nm. There are innumerable (uncountable) countless projections 3 having such a small diameter around the core portion 2.

Examples of the material for forming the magnetic fine particles 1 include iron, nickel, cobalt, iron-nickel alloy, iron-cobalt alloy, and iron-platinum alloy. In particular, the magnetic fine particle 1 made of iron, which is a ferromagnetic material, has a saturation magnetization of 1490 emu / cm ³ and a high saturation magnetization of about 90% of 1720 emu / cm ³ of bulk iron, as shown in an experimental example described later. Show. The magnetic fine particles 1 having such high magnetic properties can be made to act sensitively by a converted magnetic field (also referred to as an alternating magnetic field or a pulsed magnetic field), and can be preferably used as a magnetic fine particle for tumor cell destruction described later.

  As described above, the magnetic fine particles 1 having the beard-like protrusions 3 have not been reported so far, and can be referred to as new magnetic fine particles 1, and their uses are considered to be diverse. For example, as will be described later, when a converted magnetic field or the like is applied from the outside after being phagocytosed or endocytosed in a living cell, it causes a physical vibration motion to selectively destroy only tumor cells. It can be used as fine particles.

  In addition, when using the beard-like protrusion 3 as a magnetic separation use fine particle for drug development, or when using the beard-like protrusion 3 as a drug delivery, the physiologically active substance and the beard-like protrusion 3 It is expected that the bonding can be enhanced, so that there is an advantage that the effect can be enhanced as compared with the conventional magnetic fine particles not having the beard-like projections 3.

  Further, if the obtained magnetic fine particles 1 are applied to a magnetic fluid, it is expected that when the magnetic field is applied, the whisker-like projections 3 of the magnetic fine particles 1 are entangled and high viscosity can be obtained. As a result, it can be expected that a large viscosity change can be obtained even with a weak magnetic field, and it can be expected that a damper or the like that causes a viscosity change by the magnetic field is driven with low power.

  Further, since the obtained magnetic fine particles 1 have a very large surface area due to the presence of the beard-like projections 3, they can be expected as a magnetic particle catalyst used for the production of carbon nanotubes by the CVD method, for example. In addition, since the conventional magnetic particle catalyst is arrange | positioned two-dimensionally on the board | substrate, it was not suitable for manufacture of a large amount of carbon nanotubes.

(Manufacturing method, manufacturing equipment)
Initially, the manufacturing apparatus of the magnetic fine particle 1 is demonstrated. FIG. 5 is a schematic configuration diagram of a gas flow sputtering apparatus 10 for manufacturing the magnetic fine particles 1, and FIG. 6 is a schematic cross-sectional view showing a peripheral structure of the target holder of the magnetic fine particle 1 manufacturing apparatus 10.

  The magnetic fine particle 1 manufacturing apparatus 10 includes a vacuum vessel 15 shown in FIG. 5, and includes a hollow target 11 that generates sputter vapor 1 ′ and a sputter that introduces a sputtering gas into the hollow target 11. The gas introduction hole 13 and the generated sputtering vapor 1 ′ are transferred as a flow of sputtering gas introduced from the sputtering gas introduction hole 13 or a gas obtained by adding a carrier gas to the sputtering gas as necessary. An adjusting device (not shown) for adjusting the transfer time until the sputter vapor 1 ′ is condensed and a large number of whisker-like protrusions are generated around the magnetic fine particles 1 obtained, and a collecting unit for collecting the obtained magnetic fine particles 1 16.

  The target 11 and the sputtering gas introduction hole 13 are attached to the wall surface of the vacuum vessel 15, but are not limited to such a mode. In FIG. 5, reference numeral 17 denotes a pressure adjusting valve, and reference numeral 18 denotes a vacuum pump.

  FIG. 6 is a schematic cross-sectional view showing an example of the structure around the target holder of the magnetic fine particle manufacturing apparatus 10 (note that the example of FIG. 6 is an example and is not limited to this mode). That is, the structure around the target holder of the manufacturing apparatus 10 is pressed against the target holder 40 via the target holder 40 in which the hollow target 11 such as a cylinder is inserted, and the insulating member 21, as shown in FIG. It has at least a coupling 32 in which a sputtering gas introduction hole 13 is formed and a vacuum vessel 15 pressed against the target holder 40 via an insulating member 22 on the target exit side.

  The target holder 40 is a structure that holds the hollow target 11 such as a cylinder, and a hollow portion 41 is formed on the tube wall thereof. The cavity 41 is connected to a cooling water source (not shown) through a water supply port 42 and a drain port 43. The target holder 40 preferably has, for example, an inner diameter of 4 to 7 mm and a length of 10 to 50 mm in order to efficiently transfer the sputtering vapor generated by sputtering on the flow of the sputtering gas.

  The target holder 40 is sandwiched between the vacuum vessel 15 and the coupling 32 via the insulating members 21 and 22. Second insulating members 23 and 24 are further disposed on the side surface of the target holder 40. The second insulating members 23 and 24 are accurately meshed with the first insulating members 21 and 22 and completely insulate the target holder 40 between the vacuum vessel 15 and the coupling 32.

  A hollow target 11 such as a cylinder is attached to the inner peripheral surface of the target holder 40. The target 11 is preferably inserted in close contact with the target holder 40 in a state where, for example, an aluminum foil having a thickness of about 0.2 mm is wound around the target 11 in order to establish conduction with the target holder 40. As the target 11, those having various inner diameters can be used as long as the outer diameter matches the inner diameter of the target holder 40.

  In the coupling 32, an introduction hole 13 for the sputtering gas G is formed. On the target exit side, a cover 35 integrated with the vacuum vessel 15 is disposed as necessary. The cover 35 has a concentric opening 36 centered on the target 11, and small-diameter ejection holes 37 for blowing a carrier gas supplied as needed are provided at the periphery of the opening 36 at equal intervals in the circumferential direction. It is provided as necessary. In order to maintain the vacuum inside the apparatus, an O-ring (not shown) is interposed between the target holder 40, the vacuum vessel 15 and the coupling 32.

  In order to obtain the magnetic fine particles according to the present invention, the inside of the apparatus is evacuated to a predetermined pressure (about 1300 to 1600 Pa in an experimental example described later), and then a discharge voltage is applied to the target holder 40. Thereby, the inner surface of the target 11 is sputtered. Vapor generated by sputtering is released to the outside by the sputtering gas G from the gas introduction hole 13. At the time of sputtering, the target 11 is heated by discharge, but the target 11 expanded by heating is kept at a temperature sufficiently lower than the melting point of the target 11 because the target 11 is in close contact with the water-cooled target holder 40. Moreover, sufficient electrical contact with the target holder 40 is achieved. Therefore, it is possible to operate for a long time under stable conditions.

  The sputtering vapor released from the target 11 is sent in the direction of the recovery unit 16 as a flow along the target axis direction by the flow of the sputtering gas G (or a gas obtained by adding the carrier gas C to the sputtering gas G as required). It is. The sputtered vapor that has been sent out collides with the gas in a process in which the gas flow becomes a turbulent flow and is transferred in the direction of the recovery unit 16, thereby obtaining the magnetic fine particles 1 according to the present invention.

  The collection unit 16 is for collecting the obtained magnetic fine particles 1, and preferably includes, for example, a glass substrate or a copper mesh, but if the magnetic fine particles 1 can be collected, it is other than that. It doesn't matter. In a normal sputtering apparatus, a substrate to be formed is disposed at the position of the recovery unit 16. However, since the purpose of the present invention is not to form a deposited film, the “recovery unit 16” is provided. It is done. The collection unit 16 may be a substrate (for example, a glass substrate). At that time, the magnetic fine particles 1 are not deposited on the substrate but are collected in an “attaching” manner. The recovery unit 16 to which the magnetic fine particles 1 are attached can be obtained by taking it out from the vacuum container 15 and scraping it off from the recovery unit 16.

  In the present invention, in order for the obtained magnetic fine particles 1 to have the mustache-like projections 3, the mustache-like projections 3 are formed in the process in which the sputter vapor released from the target 11 is transferred toward the recovery unit 16. It must be. That is, as described above, the generated sputter vapor 1 ′ is transferred as a flow of the sputter gas introduced from the sputter gas introduction hole 13 (or a gas obtained by adding a carrier gas to the sputter gas as necessary), and the transfer In the process, the sputter vapor 1 'is condensed, and the transfer time until a large number of whisker-like protrusions are generated around the obtained magnetic fine particles 1 must be adjusted.

  As such a transfer time adjusting device, a pressure variable device that raises the sputtering pressure to generate a turbulent flow in the gas flow and lengthens the transfer time T from the target 11 to the recovery unit 16 to form the whisker-like projections 3, or A transfer length variable device that changes the distance S from the target 11 to the recovery unit 16 to form the mustache-like protrusion 3 can be exemplified.

  The pressure variable device is a device for changing the pressure in the vacuum vessel 15, and specifically includes a pressure adjusting valve 17. The pressure is adjusted by adjusting the exhaust capacity of the vacuum pump 18 using the pressure adjusting valve 17. When the pressure in the vacuum vessel 15 is increased by the pressure variable device, the flow of the gas flow from the target 11 toward the recovery unit 16 can be delayed. For example, by setting the pressure to 1300 to 1600 Pa and making the gas flow turbulent like Experimental Example 1 described later, the pressure up to the recovery unit 16 can be increased as compared with the case of 130 Pa (see Comparative Experimental Example 3 described later). The transfer time T can be lengthened. Specifically, the transfer time T can be set to about 5 milliseconds (Comparative Experiment 3) to about 0.3 seconds (Experiment 1). With such a pressure variable device, the sputter pressure is increased to generate a turbulent gas flow, and the transfer time T from the target 11 to the recovery unit 16 is lengthened to easily form the whisker-like projections 3 around the core unit 2. be able to.

  On the other hand, the variable transfer length device is a device for changing the position of the recovery unit 16 in the vacuum vessel 15 and changing the distance S from the target 11 to the recovery unit 16. Specifically, the recovery unit 16 is slid. A moving device can be used. When the length S from the target 11 to the recovery unit 16 is increased by the variable transfer length device, the transfer time T of the gas flow transferred from the target 11 to the recovery unit 16 can be increased. For example, by setting the length S from the target 11 to the recovery unit 16 to 500 to 700 mm as in Experimental Example 1 described later, compared to the case of a length S of about 170 mm (see Comparative Experimental Example 1 described later), Even under the same pressure condition, the transfer time T to the recovery unit 16 can be increased. Specifically, the transfer time can be about 0.05 seconds (Comparative Experiment 1) to about 0.3 seconds (Experiment 1). With such a variable transfer length device, the distance S from the target 11 to the recovery unit 16 can be changed to easily form the bearded protrusion 3 around the core unit 2.

  Next, a method for manufacturing the magnetic fine particles 1 will be described. The manufacturing method of the magnetic fine particles 1 is a process in which the magnetic fine particles 1 are manufactured by the above-described manufacturing apparatus, and the contents thereof are as described in the explanation column of the manufacturing apparatus.

  That is, the method for manufacturing the magnetic fine particles 1 will be described with reference to FIG. 5. Sputter vapor 1 ′ released from the target 11 is sputtered gas (or a gas obtained by adding a carrier gas to the sputter gas if necessary). The flow is transferred in the direction of the recovery unit 16, the sputter vapor 1 ′ is condensed in the transfer process, and the obtained magnetic fine particles 1 are recovered by the recovery unit 16. And this manufacturing method has the transfer time adjustment means which lengthens the required time T of the transfer process until a large number of whisker-like protrusions 3 are generated around the magnetic fine particles 1.

  As the transfer time adjusting means, a means (pressure variable means) for increasing the sputter pressure to generate a turbulent flow in the gas flow and extending the transfer time T from the target 11 to the recovery unit 16 to form the mustache-like protrusions 3; Alternatively, a means for changing the distance T from the target 11 to the collection unit 16 to form the mustache-like protrusion 3 (transfer length varying means) can be mentioned. Since the pressure variable means and the transfer length variable means are the same as the pressure variable apparatus and the transfer length variable apparatus in the manufacturing apparatus 10 described above, description thereof is omitted here. In the production method of the present invention, the magnetic fine particles 1 having a large number of whisker-like protrusions 3 can be easily produced by the transfer time adjusting means comprising such pressure variable means or transfer length variable means.

  FIG. 7 is a schematic view of the magnetic fine particle 1 divided into a region A in which the length L of the whisker-like projections 3 around the core portion 2 is long and a region B in which the length is short. The magnetic fine particles 1 obtained by the production apparatus and production method described above are often obtained in the form shown in FIG. Under the conditions described in Patent Document 1, fine fine particles of about 5 nm without the bearded protrusions 3 were obtained. However, although the magnetic fine particles 1 of the present invention use the same gas flow sputtering apparatus, the magnetic fine particles 1 having the beard-like protrusions 3 can be obtained by applying the transfer time adjusting device or the transfer time adjusting means as described above. It became a form. Although the reason why such a form has been made is not clear, it is caused by the process in which the whisker-like protrusions 3 grow around the core part 2 in the process in which the magnetic fine particles 1 are transferred from the target 11 toward the recovery part 16. It is considered a thing.

  A region A in which the length of the beard-like projection 3 is long is a region facing the target 11 in the process of transferring the magnetic fine particles 1, and sputter vapor and carrier gas (Ar gas or the like) supplied from the target 11 side. While the particles collide and the particles move randomly, it is considered that the beard-like projections 3 grow longer by selectively adhering to the beard-like projections 3 on the region A side. On the other hand, the region B in which the length of the beard-like projections 3 is short is considered to be a region facing the collection unit 16 in the process of transferring the magnetic fine particles 1, and the sputter vapor supplied from the target 11 side is the region. It is thought that it is shortened because it cannot go to the B side. Accordingly, the magnetic fine particles 1 obtained by the gas flow sputtering apparatus 10 are condensed into two parts, that is, a stage where the core part 2 is formed by condensation due to collision of the sputtering vapor and a stage where the whisker-like protrusions 3 are grown around the core part 2. There seems to be a stage. The present invention is characterized in that the latter stage, which has not been known so far, is specifically realized, and the characteristic magnetic fine particles 1 obtained by growing the whisker-like protrusions 3 around the core portion 2 are obtained.

  Such a form can be seen from the transmission electron micrographs shown in FIGS. 2 and 3 and can be said to be one characteristic of the magnetic fine particles 1 manufactured by the gas flow sputtering apparatus. Needless to say, it has been confirmed that if the manufacturing conditions are controlled, a true magnetic sphere or a spherical magnetic fine particle 1 other than that shown in FIG. 7 can be obtained.

[Magnetic fine particles for tumor cell destruction, cell destruction method, cell destruction device]
The present inventor has found that the magnetic fine particles 1 having the characteristic shape according to the present invention, which are easily phagocytosed or endocytosed in tumor cells, are magnetically applied by applying a conversion magnetic field from the outside, unlike spherical particles having no whisker-like projections. Discovered that it is easy to selectively destroy tumor cells by applying physical stress to microparticles to cause physical vibrational motion (including 2D motion, 3D motion, or irregular motion, the same shall apply hereinafter). . Then, it was discovered that the vibration motion of the magnetic fine particles selectively destroys or breaks only the tumor cells using the difference in vulnerability between normal cells and tumor cells.

  Conventionally, as shown in FIG. 10, the magnetic beads 50 adhere to the cell membrane 52 of the tumor cells 51 from the outside, and an external magnetic field is applied to destroy the tumor cells by applying a physical motion to the magnetic beads 50. Has been proposed (see Patent Document 2). In the same document 2, magnetic beads having a particle size of 2.8 μm and 4.5 μm are used, and the magnetic beads are attached to the cell membrane from the outside. The cell membrane was destroyed by the vibration of the beads. On the other hand, as shown in FIG. 8, the present inventor applied magnetic particles 1 phagocytosed or endocytosed in cells (in the case of simply cells, both tumor cells and normal cells). A conversion magnetic field was applied. At this time, since the magnetic fine particles 1 that have been phagocytosed or endocytosed in the cells are characteristic forms having beard-like projections 3, the magnetic fine particles 1 cause a vibrational motion different from the spherical particles due to the exchange magnetic field, I discovered that only fragile cells can be destroyed very effectively.

  What is particularly important is firstly the form of the magnetic fine particles 1 “having beard-like protrusions”. By applying a conversion magnetic field to the magnetic fine particles 1 of such a form, only vulnerable cells are extremely effective. It has been found that vibrational motion that can be destroyed is caused. Secondly, it is also important to apply the conversion magnetic field after the magnetic fine particles 1 reach the predetermined target site. In order to make the magnetic fine particles 1 reach the predetermined target site, the arrival time and the magnetic fine particles It was also found that these additional substances are important. The magnetic fine particles 1 may be phagocytosed or endocytosed in the cells, but not necessarily, and may be those adhered to the inside and outside of the cell membrane. This is because whether or not the magnetic fine particle 1 is phagocytosed or endocytosed depends on the size of the cell, the phagocytic or endocytic ability of the cell, the size of the magnetic fine particle 1, etc. It was found that the magnetic fine particles 1 having beard-like protrusions can effectively destroy fragile cells in any of the embodiments. The present invention has been made based on such a phenomenon.

  In addition, “don't eat” means to eat vagina and is used in the present application to mean that cells eat magnetic fine particles. It may also be called “Endocytosis”, and the term endocytosis is used as one of the processes in which cells take up extracellular substances. In the following column, “phagocytosis or endocytosis” is simply expressed as “phagocytosis”.

(Magnetic fine particles for tumor cell destruction)
The present inventor used the magnetic fine particles 1 having the beard-like projections 3 for tumor cell destruction, and unlike the case of Patent Document 2 to which the magnetic beads 50 shown in FIG. 10 are applied, as shown in FIG. The phenomenon in which the cells 9 phagocytose the magnetic fine particles 1 was confirmed with an electron microscope. Then, when a conversion magnetic field was applied from the outside, the phenomenon of selectively and effectively destroying only tumor cells was confirmed as compared with conventional spherical magnetic fine particles. This selective and effective destruction of tumor cells is attributed to the fact that the magnetic microparticles are provided with beard-like projections, and that the magnetic microparticles 1 caused a vibrational motion different from that of conventional spherical particles due to the conversion magnetic field. As a result, it is considered that the tumor cells were selectively and effectively destroyed. This is the gist of the present invention. The magnetic fine particles 1 here are the same as those already described with reference to FIGS. 1 to 7, and are composed of a core portion 2 and a number of mustache-like protrusions 3 around the core portion 2.

  Therefore, the magnetic fine particles 1 for destroying tumor cells are characterized by having beard-like projections 3. With such a magnetic fine particle 1, a vibration motion different from that of a conventional spherical particle can be caused by a conversion magnetic field. As long as such a behavior is exhibited, the magnetic fine particles 1 may be phagocytosed or endocytosed by the cells, or may adhere to the cell membrane, so that the size of the magnetic fine particles 1 and the length of the beard-like projections 3 are as follows. Although it is not particularly limited, in fact, cells that have been phagocytosed or endocytosed are more effective, so the size of the cell is taken into consideration, and the phagocytic or endocytic ability of the cell is also considered. Is done.

  In particular, as shown in the examples described later, the ratio of the length of the whisker-like protrusions 3 to the particle diameter D of the magnetic fine particles 1 is 5% or more and 30% or less, and the average of the particle diameters D including the beard-like protrusions 3 is The thing in the range of 100 nm or more and 300 nm or less is preferable. In addition, the eukaryotic cell is usually 10 to 100 μm, and FIG. 8 shows an embodiment in which the eukaryotic cell 9 is phagocytosing the magnetic fine particles 1 having a size of 100 nm to 300 nm. The magnetic fine particles 1 having such an average particle diameter range are phagocytosed by the cells 9 and taken into the cells.

  The magnetic fine particle 1 is configured such that the ratio of the length of the beard-like protrusion 3 to the particle diameter D is 5% or more and 30% or less. The beard-like protrusion 3 includes, as shown in FIG. In addition, a substance 5 having affinity or hindrance to a constituent protein or sugar chain antigen specifically expressed in a living cell structure can be added or adsorbed (hereinafter referred to as “addition or the like”). The magnetic fine particles 1 obtained by adding such a substance 5 to the beard-like protrusions 3 are efficiently phagocytosed and conveyed to a predetermined site after reaching the cell surface.

  Substances having affinity for such constituent proteins or sugar chain antigens include (i) antibodies against or inside the cell membrane or penetrating proteins or sugar chain antigens, or (ii) antibodies against ribosomal or lysosomal membrane antigens. Alternatively, any one selected from an affinity protein, (iii) a BAX or P53 protein against a mitochondrial membrane or a protoplasm, and (iv) an antibody or an affinity protein against an antigen distributed in the nuclear membrane can be mentioned. In addition, (i) to (iii) are, in other words, (a) an antibody against a protein in the cell membrane, an antibody against a sugar chain antigen in the cell membrane, an affinity protein for a protein in the cell membrane, a sugar chain antigen in the cell membrane. (I) antibodies against extracellular membrane proteins, antibodies against extracellular carbohydrate antigens, affinity proteins against intracellular membrane proteins, affinity proteins against intracellular carbohydrate antigens, (c) penetrates the cell membrane It can be said that the antibody against the protein that binds to the protein, the antibody against the sugar chain antigen that penetrates the cell membrane, the affinity protein for the protein that penetrates the cell membrane, and the affinity protein for the sugar chain antigen that penetrates the cell membrane.

  As an example, when the antigen is an intracellular substance and is an antigen distributed in lysosome (also referred to as lysosome) in the intracellular digestive organ, a substance having affinity for the lysosomal membrane antigen is added to the magnetic microparticle 1. By adding or the like, the magnetic fine particles 1 that have reached the cell surface are efficiently transported to intracellular lysosomes. And it becomes possible to destroy a lysosome from the inside by the magnetic fine particle 1 in a lysosome receiving the conversion magnetic field applied from the outside. Examples of substances that can be added to the magnetic fine particles 1 (substances having affinity for lysosomal membrane antigens) include LAMP (Lysosome-associated membrane protein).

  The cell destruction performance is an important factor for the time after the magnetic fine particles 1 added with the substance 5 are added to the cells. Specifically, after the magnetic fine particles 1 are added to the cells, if the magnetic fine particles 1 are phagocytosed by the cells and do not reach the lysosome, there is no effect even if a conversion magnetic field is applied from the outside. On the other hand, the time until the magnetic fine particle 1 is taken into the lysosome has tissue specificity such as phagocytic ability, endocytosis ability, intracellular movement ability and the like, and varies depending on the cell type. Therefore, it is preferable to perform a preliminary step of measuring the time until the magnetic fine particles 1 reach the lysosome using the target cell type. By performing these preliminary steps, it is possible to determine the timing for applying the conversion magnetic field.

  As another example, when the antigen is an intracellular substance and is an antigen distributed in mitochondria that is an intracellular energy producing organ, a substance 5 having affinity for the mitochondrial antigen is added to the magnetic microparticles 1 or the like. Thus, the magnetic fine particles 1 that have reached the cell surface are efficiently transported to the mitochondria in the cell. And it becomes possible to destroy a mitochondria structurally when the magnetic fine particle 1 which reached the mitochondria receives the conversion magnetic field applied from the outside. As examples of substances that can be added to the magnetic fine particles 1 (substances having affinity for mitochondrial antigens), p53 and BAX are preferable. p53 works with BAX to reduce the ability to synthesize mitochondrial adenosine triphosphate. This substance is a preferable substance for increasing the affinity of the magnetic fine particles 1 for mitochondria.

  When the magnetic fine particles 1 provided with functional proteins such as P53 and BAX reach the mitochondria, the physical action causes the mitochondrial surface to break down, and a part thereof is induced into the mitochondria through the gap to express the effect.

  By the way, if the magnetic fine particle 1 of the present invention is used, it can be efficiently transported to the mitochondria in the cell with the substance 5 having affinity for the mitochondrial antigen added. It is also possible to add to the magnetic fine particles 1 a hindering substance that inhibits the action of mitochondrial enzymes. That is, it becomes possible to damage the production structure of adenosine triphosphate, which is energy necessary for maintaining the survival function of the cell, and to destroy the cell structure. In particular, since “p53” is a substance having an action of inactivating the function of mitochondria, if it is added to the magnetic fine particles 1 to reach the mitochondria, mitochondrial damage can be enhanced regardless of the conversion magnetic field. . As described above, BAX acts as an affinity substance for mitochondria to promote physical injury by promoting viscosity with the magnetic fine particles 1.

  The cell destruction performance or intracellular adenosine triphosphate production inhibition performance is an important factor as in the case of the lysosome described above, the time after the magnetic fine particles 1 added with the substance 5 or the like are added to the cells. Specifically, after the magnetic fine particles 1 are added to the cells, if the magnetic fine particles 1 are phagocytosed by the cells and do not reach the mitochondria, there is no effect even if a conversion magnetic field is applied from the outside. On the other hand, the time required for the magnetic fine particles 1 to reach the mitochondria has tissue specificity such as phagocytic ability, endocytosis ability and intracellular movement ability, and varies depending on the cell type. Therefore, it is preferable to perform a preliminary step of measuring the time until the magnetic fine particles 1 reach the mitochondria using the target cell type. By performing these preliminary steps, it is possible to determine the timing for applying the conversion magnetic field.

  As another example, when the antigen is an intracellular substance and is an antigen distributed in the nuclear membrane which is an organ for storing intracellular protein synthesis information, a substance 5 having affinity for the nuclear membrane antigen is added to the magnetic fine particles 1. The magnetic fine particles 1 that have reached the cell surface are efficiently transported to the intracellular nuclear membrane surface. When the magnetic fine particles 1 that have reached the nuclear film receive a conversion magnetic field applied from the outside, the nuclear film can be structurally destroyed. In addition, by selecting the substance, the function of the nuclear membrane can be inhibited and the nuclear membrane can be broken.

  The nuclear membrane breakdown performance is an important factor for the time after adding the magnetic fine particles 1 added with the substance 5 to the cells. Specifically, after the magnetic fine particles 1 are added to the cells, if the magnetic fine particles 1 are phagocytosed by the cells and do not reach the nuclear membrane, applying a conversion magnetic field from the outside has no effect. On the other hand, the time required for the magnetic fine particles 1 to reach the nuclear membrane has phagocytic ability, endocytosis ability, intracellular mobility ability, tissue specificity, and varies depending on the cell type. Therefore, it is preferable to perform a preliminary step of measuring the time until the magnetic fine particles 1 reach the nuclear membrane using the target cell type. By performing these preliminary steps, it is possible to determine the timing for applying the conversion magnetic field. In addition, the effect of the nuclear envelope breakdown can be confirmed by confirming that protein synthesis in the ribosome in the cell is inhibited.

  As described above, the effects of the magnetic fine particles 1 are recognized for lysosomes, mitochondria, and nuclear membranes. However, as shown in the examples described later, the cell destruction performance of the magnetic fine particles 1 is not limited to lysosomes. The target example showed the greatest effect. The cell destruction performance at this time is considered to be caused by characteristic vibrational motion with respect to the converted magnetic field applied from the outside, as compared with the case of spherical particles. As shown in FIG. 7, such characteristic vibration motion is expected to be due to the effective action of the shape of the magnetic fine particle 1 according to the present invention having the long region A and the short region B of the beard-like projections 3. The

  Although the present application is characterized by using the magnetic fine particles 1 according to the present invention, for example, various antibodies and various antigens listed in paragraphs 0013 to 0015 of Patent Document 2 are also included in the present invention. It is possible to apply.

(Cell destruction method, cell destruction device)
In the cell destruction method according to the present invention, a step of phagocytosing magnetic fine particles 1 composed of a core portion 2 and a number of whisker-like projections 3 around the core portion 2 and applying a converted magnetic field from the outside. Applying a physical stress to the cells to selectively destroy target cells to be destroyed among the cells.

  The cell disruption device according to the present invention comprises a core portion 2 and a number of beard-like projections 3 around the core portion 2, and is phagocytosed in the cell and receives a converted magnetic field to cause physical stress on the cell. And a magnetic conversion device for selectively destroying target cells to be destroyed among the cells by applying physical stress to the magnetic fine particles 1 after being phagocytosed in the cells by a conversion magnetic field, At least.

  As explained above, the magnetic fine particles 1 that have been phagocytosed or endocytosed in the cells are different from the spherical particles having no whisker-like projections, and are physically vibrated when a converted magnetic field is applied from the outside. It becomes possible to selectively destroy vulnerable tumor cells among the cells. Further, for example, when a magnetic particle 1 capable of adding a substance 5 having an affinity for an antigen to a large number of beard-like protrusions 3 is used and the magnetic particle 1 is phagocytosed in a cell, a conversion magnetic field is applied from the outside. By applying and applying physical stress to the magnetic fine particles, it is possible to selectively destroy the target site (target cell) to be destroyed among the cells. The target cells at this time are fragile tumor cells, and it has been confirmed that healthy normal cells are not destroyed even when a similar conversion magnetic field is applied.

  Here, the magnetic field to be applied will be described. In the present invention, the conversion magnetic field is applied to the magnetic fine particles as the applied magnetic field. The conversion magnetic field is used in the latter meaning of “stable magnetic field” and “conversion magnetic field” used in MRCT (Magnetic Resonance Computer Tomography), and can be substantially expressed as a pulse magnetic field or an alternating magnetic field. The pulse magnetic field may be a single pulse or a multi-pulse. Particularly preferred is a magnetic field in which the direction in which the magnetic field is applied varies irregularly.

  In the present invention, the effect of being able to destroy the tumor cells of the present invention by repeating the movement of the magnetic microparticles 1 phagocytosed in the cells by such a conversion magnetic field, preferably by repeating the oscillating movement in multiple directions, is more effective. It can be further promoted. In particular, since the magnetic fine particles 1 according to the present invention have beard-like protrusions, unlike the spherical particles without the beard-like protrusions, the magnetic fine particles 1 are characterized in that they tend to cause two-dimensional motion, three-dimensional motion, or irregular motion by a converted magnetic field. . In particular, in the magnetic fine particle 1 having the long region A and the short region B of the beard-like projections 3 as shown in FIG. 7, such movement is likely to occur, and selective and effective destruction is likely to occur. On the other hand, it has been confirmed that conventional spherical particles may simply rotate on the spot even when a conversion magnetic field is applied, and that selective and effective destruction of tumor cells is difficult to occur.

(Treatment device)
The treatment apparatus according to the present invention includes at least the cell destruction apparatus according to the present invention, and is configured to selectively destroy tumor cells by controlling an applied magnetic field condition of a conversion magnetic field apparatus included in the cell destruction apparatus. . This treatment device can selectively destroy tumor cells only by controlling the applied magnetic field conditions of the conversion magnetic field device provided in the cell destruction device.

  Various conditions can be set and adjusted as magnetic field application conditions, and more efficient selective destruction of tumor cells can be realized.

  Hereinafter, the present invention will be described in more detail with reference to experimental examples and comparative experimental examples.

[Production experiment of magnetic fine particles]
[Experimental Example 1]
Magnetic fine particles 1 according to the present invention were produced. A gas flow sputtering apparatus 10 shown in FIG. 5 was used, and a pure iron cylindrical target (target length: 35 mm, target inner diameter: 5 mm) was used as the target 11. A copper mesh was used as the collection unit 16, and the length S from the target 11 to the collection unit 16 was 700 mm. After setting the preliminary exhaust pressure to 4 × 10 ⁻⁴ Pa, Ar gas (flow rate: 500 sccm) as a sputtering gas was fed from the sputtering gas introduction hole 13, and then the atmospheric pressure was adjusted so that the sputtering pressure was 1300 Pa. Then, gas flow sputtering was performed by applying a discharge voltage of about 300 V and a discharge current of 1.0 A to the target 11.

  Sputtering was performed for about 20 minutes to obtain the magnetic fine particles 1 on the copper mesh of the collection unit 16. When the magnetic fine particles 1 were observed with a transmission electron microscope, magnetic fine particles 1 having the forms shown in FIGS. 2 to 4 were obtained. The sputter vapor at this time arrived slowly while riding on the turbulent flow from the target 11 to the recovery unit 16. The speed was about 3 m / sec. At this time, the transfer time from the target 11 to the recovery unit 16 was about 0.3 seconds.

  The particle size distribution of the obtained magnetic fine particles 1 was measured with a transmission electron microscope and shown in FIG. As shown in the figure, the obtained magnetic fine particles 1 had a minimum particle size of 100 nm, a maximum particle size of 360 nm, and an arithmetic average particle size of 206 nm. Table 1 shows an example of the results obtained by measuring the length L of the whisker-like protrusions 3 of the magnetic fine particles 1 from a transmission electron microscope image (n = 6).

Further, when the magnetic properties of the obtained magnetic fine particles 1 were determined from a magnetization curve measured with a vibrating sample magnetometer, the saturation magnetization was 1490 emu / cm ³ (190 emu / g), and ferromagnetic particles having high saturation magnetization. It can be said that. The iron bulk saturation magnetization is 1720 emu / cm ³ (219 emu / g).

[Experiment 2]
In Experiment 1, the length S from the target 11 to the recovery unit 16 is set to 500 mm, and the transfer time from the target 11 to the recovery unit 16 is changed. Fine particles 1 were produced.

[Experiment 3]
The magnetic fine particles 1 of Experimental Example 3 were produced in the same manner as in Experimental Example 1, except that the sputtering time pressure was 1600 Pa and the transfer time from the target 11 to the recovery unit 16 was changed.

[Experimental Example 4]
Magnetic fine particles 1 of Experimental Example 4 were produced in the same manner as in Experimental Example 1, except that the Ar gas flow rate was 300 sccm and the transfer time from the target 11 to the recovery unit 16 was changed.

  In any of the above Experimental Examples 1 to 4 in which the transfer time was adjusted, the magnetic fine particles 1 having the beard-like protrusions 3 in the form seen in the transmission electron micrographs of FIGS. 2 to 4 could be obtained. .

[Comparative Experiment Example 1]
In Example 1, the length S from the target 11 to the recovery unit 16 was set to 170 mm, and the transfer time from the target 11 to the recovery unit 16 was changed. Magnetic fine particles were prepared. At this time, the transfer time from the target 11 to the recovery unit 16 was about 0.05 seconds. The obtained magnetic fine particles had an average particle diameter D of about 80 nm, and the magnetic fine particles had no whisker-like projections.

[Comparative Experiment Example 2]
In Example 1, the length S from the target 11 to the recovery unit 16 was set to 300 mm, and the transfer time from the target 11 to the recovery unit 16 was changed. Magnetic fine particles were prepared. The obtained magnetic fine particles had an average particle diameter D of about 110 nm, and the magnetic fine particles had no whisker-like projections in the same length range as the magnetic fine particles according to the present invention.

[Comparative Experimental Example 3]
Magnetic fine particles of Comparative Experimental Example 2 were produced in the same manner as in Experimental Example 1, except that the sputtering time was 130 Pa and the transfer time from the target 11 to the recovery unit 16 was changed. At this time, the sputter vapor was transferred from the target 11 to the recovery unit 16 at a high speed (jet shape) of about 100 m / sec. At this time, the transfer time from the target 11 to the recovery unit 16 was about 5 milliseconds. The obtained magnetic fine particles had an average particle diameter D of about 40 nm, and the magnetic fine particles did not have bearded protrusions.

[Comparative Experimental Example 4]
Magnetic fine particles were produced under the conditions of the example described in Patent Document 1. A gas flow sputtering apparatus 10 shown in FIG. 5 was used, and a pure iron cylindrical target (target length: 40 mm, target inner diameter: 7 mm) was used as the target 11. A transmission electron microscope observation grid was used as the collection unit 16, and the length S from the target 11 to the collection unit 16 was 70 mm. After setting the preliminary exhaust pressure to 4 × 10 ⁻⁴ Pa, Ar gas (flow rate: 200 sccm) as a sputtering gas was fed from the sputtering gas introduction hole 13, and then the atmospheric pressure was adjusted so that the sputtering pressure was 300 Pa. Then, gas flow sputtering was performed by applying a discharge voltage of 300 V and a discharge current of 1.5 A to the target 11. At this time, He gas is blown onto the target 11 as the carrier gas C from the ejection hole 37 (see FIG. 6) at a flow rate of 750 sccm, and the generated sputter vapor is discharged from the target 11 on the jet flow and passes through the opening 36. It led on the collection | recovery part 16, and was deposited.

  Sputtering was performed for about 2 seconds to obtain magnetic fine particles on a transmission electron microscope observation grid of the collection unit 16. The obtained magnetic fine particles had an average particle diameter D of about 5 nm, and the magnetic fine particles had no whisker-like projections.

[Destruction experiment of tumor cells with magnetic particles]
[Comparative Experimental Example 5]
Spherical magnetic particles having an average particle size of 50 nm (Miltenyi Biotec GmbH, Germany), 2800 nm (DYNAL Biotech ASA, Norway) and 4500 nm (DYNAL Biotech ASA, Norway) without bearded protrusions were used. When 50 nm magnetic particles are used, the used cells are DLD-1 (colorectal cancer-derived cells), and when 2800 nm magnetic particles are used, the used cells are LS-180 (colorectal cancer-derived cells) and 4500 nm. When magnetic particles were used, the cells used were HT-29 (colorectal cancer-derived cells). Each magnetic particle was put into a tumor cell and irradiated with a rapid conversion magnetic field using MRS1000 (The Magstim Company Ltd., UK) to verify the antitumor effect. The MRS 1000 generates a biphasic magnetic field and can generate a large mechanical moment with a small energy with respect to the magnetic fine particles as compared with a single-shot single-phase magnetic field generating coil. As the magnetic field conditions, the frequency was 1 Hz and 2 Hz, the magnetic field was in the range of 0.5 to 2T, and the application time was arbitrarily set in the range of 30 to 120 minutes. The results were confirmed with a fluorescence microscope, an optical microscope and an electron microscope.

(result)
An embodiment in the case of using 4500 nm magnetic particles is schematically shown in FIG. As shown in the figure, 4500 nm magnetic particles adhered to the cell membrane, and the magnetic particles were moved by the subsequent application of a conversion magnetic field to destroy the cell membrane. The same was true for 2800 nm. On the other hand, it was confirmed that the 50 nm magnetic particles were endocytosed into the cells and were vibrated by the subsequent magnetic field to destroy the cells from the inside.

[Comparative Experimental Example 6]
In Comparative Experimental Example 5, spherical magnetic particles having an average particle diameter of 300 nm (manufactured by Ademtech SA, France) having no bearded protrusions were further added, and four types of magnetic particles were used. The four types of magnetic particles are gastric cancer-derived cells (AGS, KATO-III), colon cancer-derived cells (DLD-1, HT-29, LS-180), pancreatic cancer-derived cells (MIA-PaCa2, ASPC-1), It injected | thrown-in to eight types of tumor cells of a liver origin cell (Hep-G2), and the rapid conversion magnetic field was irradiated like the comparative experiment example 5, and the antitumor effect was verified.

(result)
FIG. 12 shows the results of the antitumor effect on each tumor cell when four types of magnetic particles are used. As shown in the figure, the liver-derived cells (Hep-G2) are larger in size than other tumor cells, and thus it is considered that large magnetic particles (2800 nm, 4500 nm) were more convenient for destruction. For other tumor cells, 300 nm magnetic particles gave excellent destruction results.

[Experimental Example 5]
The magnetic fine particles 1 obtained in Experimental Example 1 were used. Otherwise, the antitumor effect was verified in the same manner as in Comparative Experimental Examples 5 and 6. As a result, when compared with the 300 nm magnetic particles of Comparative Experimental Example 6 in the same row, the ratio was 1.2 to 2.5 times, and it was confirmed that an excellent antitumor effect was obtained.

[Experimental Example 6]
The magnetic fine particles 1 obtained in Experimental Example 1 were used. The magnetic fine particle 1 was coated as a first test particle with protein A having affinity for human epithelium-related antigen on the cell membrane. Instead of the first test particles, anti-lysosomes labeled with anti-human LAMP Ab having affinity for the LAMP antigen on the lysosomal membrane were used as the second test particles. The first and second test particles were respectively added to the same 8 types of tumor cells as in Comparative Experimental Example 6, and irradiated with a rapid conversion magnetic field in the same manner as in Comparative Experimental Example 5 to verify the antitumor effect. Otherwise, the antitumor effect was verified in the same manner as in Comparative Experimental Examples 5 and 6.

  In this experimental example, for the cell membrane, human epithelial antigen that is commonly expressed in epithelial cells was targeted, and cytokeratin 8 was selected as a target for the intracellular skeleton. For endosomes, a plain magnetic material without antibodies was used as an endosome-targeted magnetic material because the target can be destroyed by irradiating the magnetic field in the process in which the tumor particles are taken up as endocytosis with the magnetic fine particles 1 as a foreign substance.

(result)
In the tumor cell membrane target treatment performed using the first test particles, an antitumor effect of 30% was confirmed. The anti-tumor effect of 50% was confirmed by the lysosomal membrane target treatment performed using the second test particles. All were able to confirm an excellent antitumor effect. Moreover, it was able to prove morphologically that the antitumor effect was derived from physical disruption based on the magnetic microparticles 1 phagocytosed in tumor cells by electron microscope observation. According to this experiment, the anti-tumor effect shows that (1) the size and shape of the magnetic microparticles 1 engulfed by the tumor cells (whisker-like projections), (2) the strength of the projected magnetic field lines, and (3) It was found to be affected by the contact time before magnetic field irradiation. It was also found that there was a difference in the antitumor effect even with the target tumor cell type.

  From the above results, the following can be said. (1) When a conversion magnetic field was applied to a cell line surface antigen as a target, cell disruption was possible. (2) It was possible to induce cell membrane disruption and cell disruption by disrupting endosomes without a cell line surface antigen target. (3) Cytotoxicity due to endocytosis was confirmed by electron microscopy. (4) It was suggested that the use of LAMP for targeting to lysosome, which is an endosomal terminal, may contribute more to cell line breakdown. (5) It was confirmed that it was possible to destroy tumor cells using a magnetized HEA antibody. (6) Regarding the antitumor effect of this magnetic field irradiation, it was considered necessary to determine the magnetic field conditions by changing the output, not the wavelength of the electromagnetic wave. (7) It was confirmed that the magnetic fine particles of Experimental Example 5 and the magnetic fine particles of Comparative Experimental Example 7 were endocytosed and finally accumulated in lysosomes, but 2.8 μm and 4.5 μm magnetic beads were deposited on the cell membrane. Although it exists, it did not exist in the lysosome. (8) When 2.8 μm and 4.5 μm magnetic beads are used, a sufficient anti-tumor effect cannot be obtained. The magnetic beads occupy 50% of the cell volume, so that cell destruction does not occur and the cells themselves The possibility of staying only in the swaying effect was considered. (9) It was suggested that magnetic beads exceeding 2.8 μm may have an effect on intracellular lysosomes.

DESCRIPTION OF SYMBOLS 1 Magnetic fine particle 1 'Sputtering vapor | steam 2 Core part 3 Beard-like protrusion 5 Antibody adhering to beard-like protrusion 8 Cell membrane 9 Cell 10 Manufacturing apparatus 11 Target 13 Sputtering gas introduction hole 15 Vacuum container 16 Collection | recovery part 17 Pressure adjustment valve 18 Vacuum pump 21, 22 First insulating member 23, 24 Second insulating member 32 Coupling 35 Cover 36 Opening portion 37 Ejection hole 40 Target holder 41 Cavity portion 42 Water supply port 43 Drainage port

G Sputtering gas C Carrier gas D Diameter of magnetic fine particle d Core diameter L Length of bearded protrusion A Area with long bearded protrusion length B Area with short bearded protrusion length S Length from target to recovery part

Claims (17)

  It consists of a core part and many beard-like protrusions around the core part, and the ratio of the length of the beard-like protrusions to the particle diameter including the beard-like protrusions is 5% or more and 30% or less. Magnetic fine particles.   2. The magnetic fine particle according to claim 1, wherein an average particle diameter including the beard-like protrusions is in a range of 100 nm to 300 nm.   The magnetic fine particle according to claim 1 or 2, wherein the length of the beard-like protrusion around the core portion is divided into a long region and a short region.   The magnetic fine particles according to any one of claims 1 to 3, which are iron fine particles formed by a gas flow sputtering method. The sputter vapor released from the target is transferred as a flow of a sputter gas or a gas obtained by adding a carrier gas to the sputter gas as necessary, and the sputter vapor is condensed in the transfer process, and the obtained magnetic fine particles are collected. A method for producing magnetic fine particles collected in
A method for producing magnetic fine particles, characterized by comprising transfer time adjusting means for lengthening the time T required for the transfer process until a large number of whisker-like protrusions are formed around the magnetic fine particles.   The transfer time adjusting means is a means for increasing the sputtering pressure to generate a turbulent flow in the gas flow and lengthening the transfer time from the target to the recovery part to form a mustache-like projection, or the distance from the target to the recovery part The method for producing magnetic fine particles according to claim 5, which is a means for forming beard-like protrusions by changing the thickness of the protrusion.   A hollow target for generating sputtering vapor, a sputtering gas introduction hole for introducing a sputtering gas into the hollow target, a sputtering gas introduced from the sputtering gas introduction hole, or as necessary. An adjusting device for transferring the gas as a gas flow obtained by adding a carrier gas to the sputtering gas, condensing the sputtering vapor in the transfer process, and adjusting the transfer time until a large number of whisker-like protrusions are formed around the magnetic fine particles obtained; And a collection unit that collects the magnetic fine particles.   The adjusting device raises the sputtering pressure to cause turbulence in the gas flow, lengthens the transfer time from the target to the recovery unit, and forms the mustache-like protrusions, or from the target The apparatus for producing magnetic fine particles according to claim 7, which is a variable transfer length device that changes the distance to the collection unit to form the beard-like projections.   Tumor cell destruction characterized by comprising a core part and a number of whiskers around the core part, and phagocytosing or endocytosing in the tumor cell to destroy the tumor cell by a conversion magnetic field applied from the outside Magnetic fine particles.   The ratio of the length of the whisker-like protrusions to the particle diameter including the whisker-like protrusions is 5% to 30%, and the average particle diameter including the whisker-like protrusions is in the range of 100 nm to 300 nm. Item 10. Magnetic fine particles for tumor cell destruction according to Item 9.   10. The tumor cell destruction according to claim 9, wherein a substance having an affinity or a hindrance to a constituent protein or a sugar chain antigen specifically expressed in a living cell structure is added to the beard-like process. Magnetic fine particles.   The substance having affinity for the constituent protein or sugar chain antigen is (i) an antibody or affinity protein for the protein or sugar chain antigen inside or outside the cell membrane, or (ii) an antibody against the ribosomal membrane antigen or lysosomal membrane antigen, or The affinity protein, (iii) a BAX or P53 protein against a mitochondrial membrane or a protoplasm, and (iv) an antibody or an affinity protein against an antigen distributed in the nuclear membrane. Fine particles for tumor cell destruction.   A step of phagocytosing or endocytosing magnetic fine particles comprising a core part and a number of whisker-like projections around the core part, and applying a conversion magnetic field from the outside to apply physical stress to the cell. And selectively destroying target cells to be destroyed among the cells.   The ratio of the length of the whisker-like protrusions to the particle diameter including the whisker-like protrusions is 5% to 30%, and the average particle diameter including the whisker-like protrusions is in the range of 100 nm to 300 nm. Item 14. The cell disruption method according to Item 13.   A magnetic fine particle comprising a core portion and a number of whisker-like protrusions around the core portion, phagocytosed or endocytosed in the cell, and subjected to a conversion magnetic field to apply physical stress to the cell; A conversion magnetic field device that applies physical stress to the magnetic fine particles after phagocytosis or endocytosis with a conversion magnetic field and selectively destroys target cells to be destroyed among the cells, Cell destruction device.   The ratio of the length of the whisker-like protrusions to the particle diameter including the whisker-like protrusions is 5% to 30%, and the average particle diameter including the whisker-like protrusions is in the range of 100 nm to 300 nm. Item 15. The cell disruption device according to Item 15.   A therapeutic apparatus comprising at least the cell disruption apparatus according to claim 15 or 16, and selectively destroying tumor cells by controlling an applied magnetic field condition of a conversion magnetic field apparatus included in the cell disruption apparatus.

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