JP2022080320A - Biopolymer operating device and biopolymer operating method - Google Patents

Abstract

To provide a new technique that has low cytotoxicity and can control polymerizable proteins only in arbitrary cell groups and intracellular regions.SOLUTION: Provided is a method for manipulating a biopolymer. The method irradiates the interface of a liquid with terahertz pulsed light to manipulate a biopolymer in the liquid by shock wave in the liquid generated by the irradiation. The biopolymer is, for example, a polymerizable protein, such as actin. In the method, the fiber structure of the polymerizable protein may be cleaved by irradiation with terahertz pulse light.SELECTED DRAWING: Figure 4

Description

Application for application of Article 30, Paragraph 2 of the Patent Act [Publications, etc.] Publication date November 12, 1st Reiwa Website address 1-1. https: // arxiv. org / abs / 1911.04674 1-2. https: // arxiv. org / pdf / 1911.04674 [Publications, etc.] Publication date November 18, 1st Reiwa Website address 2-1. https: // www. biorxiv. org / content / 10.1101 / 846295v2 2-2. https: // www. biorxiv. org / content / 10.1101 / 846295v2. full. pdf [Publications, etc.] Publication date January 20, 2nd year Website address https: // confit. atlas. jp / guide / event / lsj40 / top [Publications, etc.] Meeting name Laser Society of Japan Academic Lecture 40th Annual Meeting Date Reiwa January 20, 2 [Publications, etc.] Publication "2019 Shockwave Symposium" Proceedings (CD-ROM) Publication date February 17, 2nd year of Reiwa [Publications, etc.] Publication date February 28th, 2nd year of Reiwa Website address https: / meeting. jsap. or. jp / jsap2020s / [Publications, etc.] Publication date June 2, 2nd year Website address https: // www. nature. com / articles / s41598-020-65955-5 [Publications, etc.] Publication date June 2, 2nd Reiwa Website address https: // www. riken. jp / press / 2020/20200602_2 / index. html [Publications, etc.] Publication date July 16, 2nd year Website address 9-1. https: // www. sankeibiz. jp / businesss / news / 200716 / cpc200715000001-n1. http 9-2. https: // www. sankeibiz. jp / businesss / news / 200716 / cpc20071500010-12. http [Publications, etc.] Publication date August 26, 2nd year Website address https: // confit. atlas. jp / guide / event / jsap2020a / top

Application for application of Article 30, Paragraph 2 of the Patent Law [Publications, etc.] Meeting name 2020 (Reiwa 2nd year) Japan Society of Applied Physics Autumn Academic Lecture (Online) Date: September 8th, 2nd year of Reiwa [Publications, etc.] ] Publication date September 28, 2nd year Website address https: // www. ile. osaka-u. ac. jp / ja / wp-content / uploads / 2020/10/2009229-02_YamazakiShota_RIKEN. pdf [Publications, etc.] Meeting Name Optical / Quantum Beam Science Joint Symposium 2020 (Online) Date September 29, 2 [Publications, etc.] Meeting Name Optical / Quantum Beam Science Joint Symposium 2020 (Online) Date September 29, 2nd year [Publications, etc.] Publication date October 28, 2nd year Website address https: // www. ncbi. nlm. nih. gov / pmc / artles / PMC759516 / [Publications, etc.] Publication date October 28, 2nd year Website address https://www. qst. go. jp / site / press / 45262. html

The present invention relates to a biopolymer manipulation technique, and more particularly to a technique for cleaving a polymerized structure of a protein.

Polymerizable proteins exist in the living body in two forms: monomers and polymerized fibers. In particular, actin fibers play an essential role in cell proliferation, migration, differentiation, and development. The above-mentioned biological functions are constantly activated in cancer cells, and therefore new cancer therapeutic techniques targeting inhibition of actin fiber formation are being sought.

In the conventional method of inhibiting the formation of actin fibers, administration of a drug that binds to actin and inhibits fiber elongation (cytochalasin D, latrancurin B, micarolide B, etc.) is the mainstream, and it is actually based on drug treatment from in vitro analysis. It has been reported that it has an effect of suppressing cell division, migration and infiltration (Non-Patent Documents 1 and 2).

On the other hand, these drugs also inhibit actin fibrosis in normal cells, it is difficult to deliver drugs limited to disease areas in the body, and they are stabilized in an actin-bound state and are not excreted from cells and show high cytotoxicity. Exists as a problem. This property is due to the origin of these agents being mushroom and sponge toxins, making reversible and transient actin fiber manipulation difficult.

Saito, S., Watabe, S., Ozaki, H., Fusetani, N. & Karaki, H. Mycalolide B, a novel actin depolymerizing agent. J. Biol. Chem. 269, 29710-29714 (1994). Wakatsuki, T., Schwab, B., Thompson, N.C. & Elson, E.L. Effects of cytochalasin D and latrunculin B on mechanical properties of cells. J. Cell Sci. 114, 1025-1036 (2001).

In view of the above circumstances, it is an object of the present invention to provide a new technique having low cytotoxicity and capable of controlling a polymerizable protein only in an arbitrary cell group and intracellular region.

The present inventors have discovered that when a liquid is irradiated with terahertz pulsed light, a shock wave is generated at the liquid interface, and the energy of the terahertz light propagates in the liquid as a shock wave. Furthermore, it was discovered that this shock wave can manipulate polymerizable proteins.

The first aspect of the present invention is a biopolymer manipulation method, in which a terahertz pulse light is irradiated to the interface of a liquid, and the biopolymer in the liquid is manipulated by a shock wave in the liquid generated by the irradiation. , Characterized by that. The biopolymer is, for example, a polymerizable protein such as actin, and the fiber structure of the polymerizable protein can be cut by a shock wave generated by irradiation with terahertz pulse light. Irradiating the terahertz pulse light to the liquid interface means that in addition to directly irradiating the terahertz pulse light to the liquid interface, if the light energy can be propagated to the liquid interface as terahertz pulse light, the film, glass, etc. It means irradiating the liquid interface with terahertz pulsed light through the living tissue. According to the present invention, the fiber structure can be cut not only at the tip of the fiber structure but also in the middle of the structure. Further, the cut region can be limited to a specific region by adjusting the irradiation position of the terahertz pulse light. In addition, irradiation with terahertz pulse light does not cause protein denaturation, aggregation, or cell damage, which is advantageous in terms of safety.

In this embodiment, the glass plate holding the biopolymer may be arranged at a position separated from the bottom surface of the film bottom dish containing the liquid by a predetermined distance, and the bottom surface of the film bottom dish may be irradiated with the terahertz pulse light. .. It is desirable that the above-mentioned predetermined distance is a distance so far that the thermal effect of the irradiation of the terahertz pulse light does not reach, and the distance is such that the energy can be reached by the shock wave generated in the liquid. Biopolymers may be present in living cells. For example, cells may be cultured on a glass plate and irradiated with terahertz pulse light as described above. Alternatively, the biopolymer may be dissolved in the liquid.

The second aspect of the present invention is a biopolymer manipulation device, which holds a liquid and holds a biopolymer at least at a position away from the interface of the liquid, and terahertz pulse light at the interface of the liquid. It is characterized by comprising a light irradiation unit for irradiating the biopolymer, and manipulating the biopolymer by a shock wave in the liquid generated by irradiation with the terahertz pulse light.

According to the present invention, the cytotoxicity is low, and the polymerizable protein can be controlled only in an arbitrary cell group and intracellular region.

The figure which shows the outline structure of the biopolymer operation apparatus which concerns on Example 1. FIG. The figure which shows the experimental result which concerns on Example 1. The figure which visualized the shock wave generated by the terahertz wave irradiating the liquid surface. The figure which shows the outline structure (excluding the optical system part) of the biopolymer operation apparatus which concerns on Example 2. FIG. The figure which shows the experimental result which concerns on Example 2.

Hereinafter, embodiments for carrying out the present invention will be described with reference to the drawings, but the present invention is not limited thereto. The components of each embodiment described below can be combined as appropriate.

[1. Irradiation of actin in an aqueous solution with terahertz waves]
First, the effect of terahertz light irradiation on the formation of actin fibers in an aqueous solution was investigated. FIG. 1 is a diagram showing an outline configuration of an actin fiber cutting device (biopolymer operating device) 1 according to the present embodiment. The actin fiber cutting device 1 includes a light irradiation unit 10 that irradiates a terahertz pulse wave, an attenuator 11 for adjusting the intensity, a parabolic mirror 12, and a film bottom dish 13.

The light irradiation unit 10 includes a free electron (FEL) laser light source and is configured to be capable of irradiating pulsed light having a terahertz frequency. In this embodiment, the light irradiation unit 10 irradiates a macro pulse consisting of about 100 micropulses having a pulse width of 5 picoseconds at a repetition frequency of 5 Hz. The center frequency of the terahertz wave is 4 THz and the bandwidth is 1 THz. The terahertz light was loosely focused by the parabolic mirror 12 and the focal point was 25 mm away from the sample. The beam diameter is 4 mm on the sample. The beam intensity can be adjusted by an attenuator consisting of wire grid modulators 11a, 11b and a terahertz attenuator 11c. The beam intensity of this example was 80 to 250 μJ / cm ² per micropulse. The terahertz pulse light from the light irradiation unit 10 irradiates the film unit of the film bottom dish 13. Although the terahertz pulse light is applied to the film portion, the film portion is sufficiently thin and the light energy of the terahertz pass light propagates to the interface between the film portion and the actin aqueous solution 14, so that the above irradiation is performed at the interface of the actin aqueous solution 14. It can be regarded as the irradiation of terahertz pulse light to.

The actin aqueous solution 14 is obtained by adding a buffer solution for polymerizing actin to a solution of G-actin (spherical actin) obtained by dissolving purified actin in water. The actin aqueous solution 14 was dropped onto the film bottom dish 13 so as to have a thickness of 1 mm, and a polymerization reaction was carried out on the film bottom dish 13.

From the bottom of the film bottom dish 13, pulsed light having an intensity of 80 μJ / cm ² and a wavelength of 4 THz was irradiated for 30 minutes. In addition, for comparison, an actin aqueous solution not irradiated with terahertz pulse light was placed next to the actin aqueous solution irradiated with terahertz pulse light.

FIG. 2 shows the results of staining actin fibers with a fluorescent probe and observing them with a fluorescence microscope after irradiation with terahertz pulse light for 30 minutes. FIG. 2A is an observation result of a non-irradiated sample of terahertz pulse light, and FIG. 2B is an observation result of an irradiated sample of terahertz pulse light. The concentration of actin protein in the field of view under the microscope is the same (2.4 μM), and only the actin fibers are stained and visualized. Obviously, the irradiated sample (FIG. 2 (B)) has a lower luminance than the non-irradiated sample (FIG. 2 (A)) and has a smaller number of actin fibers having a detectable length. I understand. Further, FIG. 2C shows the number of actin fibers counted in the field of view of the microscope, and it can be seen that the number of actin fibers is reduced by about 40% by irradiation with terahertz pulse light. Further, when one actin fiber was magnified and observed, no influence was observed on the linear normal fiber structure of the actin fiber, and it was confirmed that no structural change such as modification or aggregate formation occurred. ..

As described above, by irradiating the actin aqueous solution with terahertz pulse light, it is possible to cleave or inhibit the formation of actin fibers. In the conventional method using a drug, in addition to the inhibition of fiber elongation, the structural change of actin due to drug binding becomes a problem, but as described above, this method does not cause a structural change.

In addition, the results of this experiment using an aqueous solution system show that the inhibitory effect on fiber formation by terahertz pulse light irradiation is a direct action not mediated by intracellular phenomena (intracellular signals, chemical modifications, protein regulators). There is. In addition, the energy of the terahertz pulsed light to be irradiated is sufficiently small, about ¹⁰⁸ W / cm ² , so that side effects due to thermal action are not caused.

The thermal effect of terahertz light irradiation does not extend to the deep part of the liquid. Terahertz light directly excites intermolecular oscillations in hydrogen-bonded networks. Considering the absorption coefficient of the 4 THz wave of water, 99% of the energy is absorbed within 10 μm from the liquid surface. This strong absorption results in a rapid rise in local temperature and pressure, producing shock waves in water with high efficiency.

FIG. 3 shows the experimental results of visualizing the shock waves in the water generated in this way. In this figure, when terahertz pulse light with a frequency of 4 THz, a beam diameter of 0.7 mm, and an energy density of 4.5 mJ / cm ² per micropulse (power density of 3.1 GW / cm ² ) is gently converged on the water surface and irradiated. The underwater condition of the water is observed by the shadow graph method. From the figure, it can be seen that a plane shock wave is generated in the water. Since it is a plane wave, it can propagate over a long distance, and it can be seen that it propagates by 3 mm or more from the surface.

For the above reasons, it can be said that the operation of actin molecules by irradiation with an aqueous actin solution of terahertz pulse light is not due to the thermal effect of the terahertz wave, but to the shock wave accompanying the irradiation of the terahertz pulse light. That is, it is possible to irradiate the interface of the liquid with terahertz pulse light and manipulate the biopolymer in the liquid by the shock wave in the liquid generated by this irradiation.

It is also possible to irradiate high-intensity visible light or near-infrared light to generate a shock wave in water by plasma conversion and ablation, but since spherical waves are generated, the shock wave generation efficiency is poor, and 10 ¹⁵ Since energy of W / cm ² is required, there is a problem in terms of safety to the living body.

[2. Irradiation of actin in cells with terahertz waves]
Next, in order to investigate the effect of terahertz wave irradiation on actin in living cells, the actin fibers present in cultured human cells were irradiated with terahertz waves.

In order to investigate the effect on actin fibers in living cells, it is necessary to maintain the vital function of the cells in the irradiation environment. Therefore, the actin fiber cutting device (biopolymer manipulation device) according to this embodiment includes a portable irradiation / observation platform combining an optical system for terahertz pulse light irradiation, a cell culture device, and a fluorescence microscope, and a free electron laser light source. Constructed in combination. Since the configuration of the optical system is the same as that of the example of FIG. 1, the repeated description will be omitted.

FIG. 4 is an enlarged view of a cell culture device portion and a fluorescence microscope portion. The cell culture apparatus portion (holding portion) includes a heating stage 21 having an opening, a film bottom dish 22, a spacer 23 for adjusting the distance between the cultured cells and the bottom surface of the film bottom dish 22, and a cultured cell. It has a cover glass 24 to carry. The heating stage 21 keeps the culture solution 26 in the film bottom dish 22 at a constant temperature (for example, 37 ° C.). The spacer 23 is used to adjust the cover glass 24 to which the cultured cells are adhered so as to be arranged at a predetermined distance from the bottom surface of the film bottom dish. In the present embodiment, the predetermined distance is at least 800 μm to 2800 μm. It is adjustable in the range. Cultured cells (HeLa cells) 25 are cultured on the cover glass (sample plate) 24.

Further, in this embodiment, a fluorescence microscope (not shown) having a water immersion objective lens 27 is installed above the condensing surface in order to observe the influence of terahertz wave irradiation over time. By staining the actin fibers in the cultured cells 25 with a fluorescent probe and observing them with a fluorescence microscope, it is possible to observe the effects of irradiation over time. The fluorescence microscope can be omitted from the viewpoint of cutting actin fibers.

Cultured cells (HeLa cells) 25 were seeded in cover glass 24 2 days prior to the experiment and added to Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 ° C., 5% CO ₂ conditions. Cultured under. In addition, silicon rhodamine (SiR) actin, which is a fluorescent probe, was used for staining intracellular actin fibers. Since SiR actin has high cell membrane permeability and fluoresces when it binds to actin fibers, only intracellular actin fibers can be observed with high resolution. After staining, the cover glass 24 to which the cultured cells were adhered was placed on the film bottom dish 22. At this time, the cover glass 24 is installed at a position 800 μm away from the bottom surface of the film bottom dish 22 by the spacer 23. Then, the film bottom dish 22 was placed on the heating stage 21, and the bottom surface of the film bottom dish 22 was irradiated with terahertz pulse light while the medium was kept at 37 ° C.

In this embodiment, the irradiation conditions are the same as those in the first experiment except that the beam intensity of the terahertz wave is 250 J / cm ² per micropulse. That is, the terahertz pulse light has a repetition frequency of 5 Hz for a macro pulse consisting of about 100 micropulses having a pulse width of 5 picoseconds, the center frequency of the terahertz beam is 4 THz, and the bandwidth is 1 THz. The terahertz beam is loosely focused, the focal point is 25 mm away from the sample, and the beam diameter is 4 mm on the sample.

For comparison, observations are also made under the same conditions except that terahertz pulse light is not irradiated.

FIG. 5 is an observation result of a fluorescence microscope showing the experimental results. 5 (A) is an observation result of a non-irradiated sample of terahertz pulse light, and FIG. 5 (B) is a partially enlarged view of FIG. 5 (A). 5 (C) is an observation result of an irradiation sample of terahertz pulse light, and FIG. 5 (D) is a partially enlarged view of FIG. 5 (C). In the sample irradiated with terahertz pulse light for 30 minutes, it was confirmed that the actin fibers existing in the cells were fragmented (arrow portion in FIG. 5D). This result indicates that actin fibers in the living body are also cleaved by terahertz pulse light irradiation, similar to the above-mentioned purified actin.

When the experiment was conducted by changing the distance between the cover glass 24 and the bottom surface of the film bottom dish 22, the energy due to the irradiation of the terahertz wave reached at least a depth of 1000 μm from the interface. This means that the energy was not transmitted by the heat effect, but the irradiated photon energy was transmitted in water as a pressure wave (shock wave) and cut the actin fibers in the cell.

According to this embodiment, actin fibers can be cleaved in the middle of the structure (FIG. 5 (D)). The conventional mechanism of action of inhibiting fiber formation by drug administration is that the drug binds to and capps to the tip of the fiber to suppress the bonding of monomeric actin, and inhibits elongation only at the end. That is, the actin fiber cutting of this example is a novel effect of inducing disintegration of the fiber structure, which has never been seen before. This action suggests that terahertz light irradiation may cut and disintegrate fibers more efficiently and in a short time compared to drugs. In addition, the aggregated (modified) form of actin generated by drug administration was not observed in the cells as in the aqueous solution system, and it was confirmed that the survival rate of the cells was 99% or more. Furthermore, this method can be applied to an arbitrary region in a limited manner by condensing terahertz light. Therefore, this method can be applied only to a specific region, which has been difficult in the past, and has a great advantage in terms of safety.

[3. Other Examples]
The present invention can be regarded as a biopolymer manipulation method in which a terahertz pulse light is irradiated to the interface of a liquid and the biopolymer in the liquid is manipulated by the shock wave in the liquid generated by the irradiation. In the above explanation, actin is targeted as a biopolymer and structural manipulation is performed by irradiation with terahertz pulse light, but in addition to actin, any polymerizable protein including tubulin, keratin, amyloid, collagen, etc. is targeted. You can also do it.

Further, the terahertz light that can be used is preferably high frequency, high energy, and short pulse so as to cause a shock wave (pressure wave) in the liquid, but is not limited to the frequency and energy density described above. .. For example, the frequency of the terahertz beam may be 1 THz or more and 25 THz or less, and more preferably 4 THz or more and 20 THz or less. The energy density may be 50 μJ / cm ² or more and 500 μJ / cm ² per micropulse. The pulse width may be 1 ps (picoseconds) or more and 1000 ps or less.

Further, in the above description, a free electron laser light source is used as a light source of the terahertz beam, but the present invention is not limited to this. Recent advances in technology have proposed powerful, compact, and inexpensive light sources, and any light source that can irradiate terahertz waves satisfying the above conditions may be used. By adopting a small light source, it is possible to construct a portable biopolymer operating device.

Further, the terahertz wave may be irradiated to the living tissue instead of the cultured cells via a liquid such as water. Further, the liquid may be water in a living tissue. Since the shock wave accompanying the terahertz wave irradiation reaches a depth of about 2 to 3 mm and the thickness of the skin in the human body is about 2 mm, the influence of the terahertz pulse light reaches the deep part of the skin tissue. That is, the biopolymer manipulation device can irradiate the skin surface of a human body or an animal with terahertz pulse light, and the shock wave in the living tissue generated by this irradiation can manipulate the polymerizable protein in the deep part of the tissue. Be expected.

1: Biopolymer operation device 10: Light irradiation unit 11a, 11b: Wire grid splitter 11c: Terrahertz attenuator 12: Radial surface mirror 13: Film bottom dish 21: Heating stage 22: Film bottom dish 23: Spacer 24: Cover Glass 25: Cultured cells (HeLa cells) 25: Culture solution 27: Water-immersed objective lens

Claims (9)

A method for manipulating a biopolymer, which comprises irradiating an interface of a liquid with terahertz pulse light and manipulating the biopolymer in the liquid by a shock wave in the liquid generated by the irradiation. The biopolymer is a polymerizable protein and is
Irradiation with the terahertz pulse light cuts the fiber structure of the polymerizable protein.
The biopolymer manipulation method according to claim 1. The sample plate to which the biopolymer is adhered is placed at a position separated from the bottom surface of the film bottom dish containing the liquid by a predetermined distance, and the bottom surface of the film bottom dish is irradiated with the terahertz pulse light.
The biopolymer manipulation method according to claim 1 or 2. The frequency of the terahertz pulse light is 1 THz or more and 25 THz or less.
The biopolymer manipulation method according to any one of claims 1 to 3. The pulse width of the terahertz pulse light is 1 ps or more and 1000 ps or less.
The biopolymer manipulation method according to any one of claims 1 to 4. The energy of the terahertz pulse light is 50 μJ / cm ² or more and 500 μJ / cm ² or less.
The biopolymer manipulation method according to any one of claims 1 to 5. A holding part that holds the liquid and holds the biopolymer at least at a position away from the interface of the liquid.
A light irradiation unit that irradiates the interface of the liquid with terahertz pulse light,
Equipped with
A biopolymer manipulation apparatus comprising manipulating the biopolymer by a shock wave in the liquid generated by irradiation with the terahertz pulse light. The biopolymer is a polymerizable protein.
The biopolymer operating device according to claim 7. The holding portion includes a film bottom dish and a sample plate carrying the biopolymer.
The light irradiation unit irradiates the film portion of the film bottom dish with the terahertz pulse light.
The biopolymer manipulation apparatus according to claim 7 or 8.

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