JP2008017739A - Laser irradiation type foreign substance introduction device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a laser irradiation type foreign substance introduction device which can repeadedly give impact waves to a biological tissue without burning the tissue. <P>SOLUTION: This laser irradiation type foreign substance introduction device has a light transmission member 110 through which the laser beam irradiated from outside transmits, an impact wave-generating layer 120 which absorbs the transmitted laser beams to generate impact waves, and an impact wave transmitter 130 which transmits the impact waves to cells desired to introduce a foreign substance thereinto. By repeatedly irradiating the device with pulse laser beams, heat energy and impact waves acting to introduce a foreign matter into cells in a living body are generated in the impact wave-generating layer 120. The impact wave transmitter 130 utilizes a transmittance speed difference between the heat energy and the impact waves to transmit only impact waves to a biological tissue. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a laser irradiation type foreign substance introduction device for introducing a foreign substance into a cell by applying a shock wave induced by laser light.

  Gene therapy is a therapeutic method that may be applied not only to gene diseases but also to various intractable diseases such as cancer and AIDS. In order to succeed in gene therapy, it is essential to establish a technique for safely and efficiently introducing a target gene (DNA) into a target cell.

  Various gene transfer methods have been developed so far. For example, as a method for introducing a gene (DNA) into a cell at an in vivo level, a viral vector method using a virus vector into which the gene is introduced, nanometer-sized gold microparticles coated with DNA at high speed There are a gene gun (gene gun / particle gun) method for implanting cells, and an electroporation method for applying electrical stimulation to cells.

  The viral vector method is widely used because of its relatively high introduction efficiency, but there is a problem in safety because it uses a virus. The gene gun method has a problem that the gene (DNA) can be introduced only into the surface layer of the tissue and the introduction efficiency is low. The electroporation method is invasive because the electrode is placed at the target site, and has a problem that introduction efficiency is low. As described above, the method of introducing a gene (DNA) into cells in a living body has major problems from the viewpoint of introduction efficiency and safety.

  As a method for solving the above problem, a method of introducing a foreign substance such as DNA into cells in a living body using a laser-induced stress wave has been proposed (Non-Patent Document 1). When a solid medium is irradiated with pulsed laser light, the medium irradiated with the pulsed laser light is turned into plasma, and a strong shock wave (laser-induced stress wave) and thermal energy are generated as the medium expands. When this shock wave is applied to a cell, the cell takes in a foreign substance around it (Non-Patent Document 2).

FIG. 3 is a schematic diagram of the method of Non-Patent Document 1. First, plasmid DNA (foreign substance) is injected into the dermis of the rat dorsal skin 15, and a black rubber disc 11 to which a transparent plate 10 of polyethylene terephthalate is bonded is placed thereon. Next, the second harmonic (wavelength 532 nm, maximum laser fluence 1.9 J / cm ² ) 12 of the Q switch Nd: YAG laser 12 is irradiated to the black rubber disk 11 several times (1 to 5 times) to generate plasma. 13 and a shock wave 14 are generated, and the shock wave acts on cells in the dermis immediately below. By this method, plasmid DNA is introduced into epidermal cells with relatively high introduction efficiency (Non-Patent Documents 1 and 2).

In this way, by using laser-induced stress waves, foreign substances such as DNA can be introduced into cells in living tissue with a relatively high introduction efficiency without worrying about safety problems due to viruses.
Ogura M., Sato S., Nakanishi K., Uenoyama M., Kiyozumi T., Saito D., Ikeda T., Ashida H. and Obara M. 2004. "In Vivo Targeted Gene Transfer in Skin by the Use of Laser -Induced Stress Waves ". Lasers in Surgery and Medicine 34: 242-248. Supervised by Koji Sugioka and Akira Yabe, “Laser Micro / Nano Processing”, CM Publishing, p.334-336.

  However, in the method of Non-Patent Document 1, there is a problem in that a living tissue is burned when pulsed laser light is repeatedly irradiated in order to further increase the efficiency of introducing a foreign substance. That is, when pulsed laser light is repeatedly applied to a black rubber disc, the temperature of the black rubber disc rises, and the living tissue directly in contact with it is burned (symptoms such as erythema occur).

  In the foreign substance introduction method using laser-induced stress waves, the introduction efficiency can be improved by increasing the number of times of irradiation with pulsed laser light. However, in the method of Non-Patent Document 1, erythema is generated only by irradiating pulsed laser light several times. Therefore, the number of irradiation cannot be increased further in consideration of safety. Therefore, avoiding burns of living tissue due to irradiation with pulsed laser light is a major issue in improving the introduction efficiency of foreign substances.

  The present invention has been made in view of the above points, and a laser capable of repeatedly acting shock waves on cells in a living tissue without causing burns to the living tissue even when repeatedly irradiated with a pulsed laser beam. An object is to provide an irradiation type foreign substance introduction device.

  The laser irradiation type foreign substance introduction device of the present invention is a laser irradiation type for introducing a foreign substance into a cell by applying a shock wave induced by laser light to a cell in a living tissue into which the foreign substance is introduced in the vicinity. A foreign substance introduction device, a light transmitting body through which laser light irradiated from the outside transmits, a shock wave generating layer that generates a shock wave by absorbing the laser light transmitted through the light transmitting body, and the shock wave generating layer And a shock wave transmitting body for transmitting the shock wave generated in step 1 to the living tissue.

  According to the present invention, since the shock wave can be repeatedly acted on the cells in the living tissue without causing a burn on the living tissue, the introduction efficiency of the foreign substance can be improved.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

  FIG. 1 is a diagram showing the structure of a laser irradiation type foreign substance introduction device according to an embodiment of the present invention.

  Laser irradiation type foreign substance introduction device 100 according to the present embodiment employs a configuration in which shock wave generating layer 120 is sandwiched between light transmitting body 110 and shock wave transmitting body 130. From the viewpoint of safety and handling properties, the shock wave generating layer 120 is preferably pressure-bonded or bonded to the light transmitting body 110 and the shock wave transmitting body 130.

  The light transmitting body 110 is a member that allows laser light irradiated from the outside to pass through the shock wave generation layer 120. Therefore, the material of the light transmitting body 110 is preferably light-transmitting, and particularly preferably colorless and transparent.

  The light transmitting body 110 also has a function of increasing the pressure of the shock wave generated in the shock wave generation layer 120 by confining the plasma generated in the shock wave generation layer 120. Therefore, it is preferable that the material of the light transmitting body 110 has a strength that can withstand the plasma and shock waves generated in the shock wave generating layer 120, and even if the plasma and shock waves are repeatedly generated in the shock wave generating layer 120, they can withstand those shocks. Those having high strength are particularly preferred.

  As described above, the material of the light transmitting body 110 is not particularly limited, but a material having light transmittance and high strength is preferable. Therefore, the material of the light transmitting body 110 can be, for example, a transparent resin (polyethylene terephthalate, etc.) or a transparent inorganic material (glass, quartz, ceramics, sapphire), etc., preferably a transparent inorganic material, particularly preferably sapphire. It is. The colorless and transparent sapphire plate is particularly preferable as the light transmitting body 110 because it is not easily damaged even when repeatedly irradiated with a pulse laser having a high laser fluence.

  The thickness of the light transmitting body 110 is not particularly limited, and may be determined based on the strength of the material of the light transmitting body 110. For example, when the light transmitting body 110 is a sapphire plate, the thickness is preferably 1.0 mm or more.

  The size (length × width size) of the light transmitting body 110 is not particularly limited, but is preferably larger than the irradiation area of the laser beam, and particularly preferably larger than the size of the shock wave generating layer 120.

In a preliminary experiment conducted by the present inventor, a laser irradiation alien substance introduction device using a colorless and transparent sapphire plate having a thickness of 1.2 mm as a light transmitting material was applied to a pulsed laser beam having a laser fluence of 3 J / cm ^2. Even when irradiated with 500 times at a frequency of 10 Hz, the light transmitting body was not damaged.

  The shock wave generation layer 120 generates a shock wave and thermal energy when absorbing the laser light. The material of the shock wave generating layer 120 is not particularly limited, but a material that absorbs the irradiated laser beam and causes ablation by the energy or a property that generates plasma by the energy is preferable. For example, when the laser beam is a fundamental wave of Nd: YAG laser (wavelength 1064 nm), examples of the material of the shock wave generation layer 120 include a black pigment that absorbs near-infrared light and a black rubber plate. The reaction induced in the shock wave generation layer 120 by the irradiation of the laser light shifts from laser ablation to generation of plasma in accordance with the increase of the laser fluence. It is generated in the shock wave generation layer 120.

  The thickness of the shock wave generation layer 120 is not particularly limited, but may be, for example, 1.0 mm to 3.0 mm. If the shock wave generation layer 120 is too thin, the shock wave generation layer 120 cannot withstand the laser light irradiated during one foreign substance introduction process, and the laser light penetrates the shock wave generation layer 120. On the other hand, if the shock wave generating layer 120 is too thick, the shock wave generating layer 120 absorbs the shock wave, and the transmission efficiency of the shock wave to the living tissue is reduced.

  The size (length × width size) of the shock wave generation layer 120 is not particularly limited, but is preferably larger than the irradiation area of the laser beam.

  The shock wave transmitting body 130 is a member that transmits shock waves generated in the shock wave generating layer 120 to a living tissue. Therefore, it is preferable that the material of the shock wave transmitting body 130 is strong enough to withstand the shock wave and can propagate the shock wave. In general, since a material having high hardness can propagate a shock wave, it is particularly preferable that the material of the shock wave transmitting body 130 has high strength and high hardness.

  The shock wave transmitting body 130 also has a function of preventing thermal energy generated in the shock wave generating layer 120 from being transmitted to the living tissue. Therefore, it is preferable that the shock wave transmission body 130 has a large heat capacity (specific heat).

  Thus, the material of the shock wave transmitting body 130 is not particularly limited, but a material having high strength and high hardness and high specific heat is preferable. As what satisfy | fills this condition, a sapphire board is mentioned, for example. Since the sapphire plate has high strength and high hardness (Mohs hardness 9) and has a relatively large specific heat (0.75 kJ / kg · ° C .: 25 ° C.), it is preferable as the shock wave transmitting body 130.

  The thickness of the shock wave transmitting body 130 may be determined based on the strength of the material of the shock wave transmitting body 130 and the specific heat. For example, when the shock wave transmitting body 130 is a sapphire plate, the thickness is not particularly limited, but may be 1.0 mm to 3.0 mm. If the shock wave transmitting body 130 is too thin, it will be easily damaged by the shock wave and heat will be easily transmitted to the living tissue. Further, if the shock wave transmitting body 130 is too thick, the shock wave is difficult to be transmitted to the living tissue.

  The size (length × width size) of the shock wave transmitting body 130 is not particularly limited, but is preferably larger than the size of the shock wave generating layer 120.

In a preliminary experiment conducted by the present inventor, a laser fluence 3 J / cm ² pulse laser beam was applied to the laser irradiation type foreign substance introduction device of the present invention using a 1.0 mm-thick colorless and transparent sapphire plate as a shock wave transmitter. The shock wave transmitting body was not damaged even when irradiated with 500 times at a frequency of 10 Hz. In addition, even when a shock wave was generated under the above conditions, no burns were observed in the living tissue in contact with the shock wave transmitting body 130 (see Examples).

  Next, a procedure for introducing a foreign substance into cells in a living tissue using the laser irradiation type foreign substance introduction device according to the present invention will be described.

  FIG. 2 is a diagram for explaining a method of introducing a foreign substance using the laser irradiation type foreign substance introduction device 100 of FIG. Here, an example in which the foreign substance 180 is introduced into the cell 170 in the living tissue 160 by irradiating the laser irradiation type foreign substance introduction device 100 with the lens 140 using the lens 140 is shown.

  The “biological tissue” here is not particularly limited, and examples thereof include epithelial tissue, connective tissue, muscle tissue, nerve tissue and the like. Further, the “cell” herein is not particularly limited, and examples thereof include differentiated cells such as fibroblasts, muscle cells, nerve cells or glial cells, and undifferentiated cells such as hematopoietic stem cells or nerve stem cells. . The “foreign substance” here is not particularly limited, and examples thereof include DNA, RNA, polypeptide, protein, lipid, saccharide and the like.

  First, the foreign substance 180 is introduced in the vicinity of the cell 170. At this time, it is preferable that the foreign substance 180 is temporarily localized around the cell 170 as shown in FIG. A method for introducing the foreign substance 180 is not particularly limited. For example, a solution containing the foreign substance 180 may be injected into the living tissue 160.

  Next, the laser irradiation type foreign substance introduction device 100 is arranged on the surface of the living tissue 160. At this time, the laser irradiation type foreign substance introduction device 100 is arranged so that the shock wave transmitting body 130 contacts the living tissue 160. The place where the laser irradiation type foreign substance introduction device 100 is arranged is preferably directly above the cell 170 into which the foreign substance is introduced. A shock wave propagating agent may be applied to the contact surface between the surface of the living tissue 160 and the shock wave transmitting body 130. By applying the shock wave propagating agent, reflection of the shock wave at the contact surface between the shock wave transmitting body 130 and the living tissue 160 is suppressed, so that the shock wave can be transmitted to the living tissue 160 more efficiently. Examples of the shock wave propagating agent include commercially available ultrasonic jelly and silicon grease.

  Next, using the lens 140, the laser beam 150 is condensed and applied to the upper surface of the shock wave generation layer 120 (contact surface with the light transmission body 110), and the shock wave generation layer 120 generates a shock wave and thermal energy. The shock wave quickly propagates to the cells 170 in the living tissue 160 via the shock wave transmission body 130. On the other hand, thermal energy is prevented from propagating to the living tissue 160 by the shock wave transmission body 130.

  The type of the laser beam 150 is preferably a pulsed laser beam from the viewpoint of repeatedly generating shock waves, and a nanosecond pulsed laser beam is particularly preferable from the viewpoint of sufficiently increasing its intensity.

  The wavelength of the laser beam 150 is not particularly limited, but is preferably a wavelength that is not absorbed by the living tissue 160 from the viewpoint of safety. For example, when a foreign substance is introduced into the skin or subcutaneous tissue of a mouse or human, the wavelength of the laser beam 150 is preferably 700 nm to 1500 nm, and particularly preferably 900 nm to 1200 nm. This is because the light absorption of hemoglobin in blood and water in tissue is particularly weak in this wavelength region.

The laser fluence on the irradiation surface of the laser beam 150 (the upper surface of the shock wave generation layer 120) is not particularly limited, but is preferably as high as possible within a range in which the light transmitting body 110 is not damaged from the viewpoint of introduction efficiency. For example, if the transparent member 110 is a sapphire plate, laser fluence is preferably from ^{0.5J / cm 2 ~10J / cm 2} , 2J / cm 2 ~5J / cm 2 is particularly preferred.

  The irradiation area on the irradiation surface of the laser beam 150 (the upper surface of the shock wave generation layer 120) is not particularly limited, and may be arbitrarily determined.

  The number of times of irradiation with the pulsed laser beam 150 is not particularly limited, but may be appropriately set within a range in which the light transmitting body 110 is not damaged from the viewpoint of the amount of the foreign substance 180 introduced. For example, when the light transmission body 110 is a sapphire plate, 1 to 5000 times is preferable, and 10 to 1000 times is particularly preferable.

  During the continuous irradiation time of the pulse laser beam 150 (the time from the first pulse laser beam irradiation to the end of the last pulse laser beam irradiation in a series of irradiation), the thermal energy generated in the shock wave generation layer 120 is the living body. It is preferable to make it shorter than the time until propagation to the tissue 160. When the number of times of irradiation necessary to introduce a sufficient amount of a foreign substance is large, the continuous irradiation time can be shortened by dividing the irradiation of the pulse laser beam into a plurality of times. For example, when irradiating 5000 times, 100 continuous irradiations may be repeated 50 times with a break in between. By doing so, it is possible to prevent the thermal energy generated in the shock wave generation layer 120 from propagating to the living tissue 160 even when the number of times of irradiation with the pulse laser beam is large.

  According to the above procedure, the cell 170 that has received the shock wave temporarily changes the structure of the cell membrane and takes in the foreign substance 180 in the surrounding area.

  Thus, according to the present invention, only the shock wave among the shock wave and thermal energy generated by irradiating the laser beam can be propagated to the living tissue. Therefore, the shock wave can be repeatedly applied to the living tissue without causing a burn on the living tissue, so that the foreign substance can be safely and efficiently introduced into the cells in the living tissue.

  Hereinafter, more specific embodiments (examples) of the present invention will be described. In addition, this invention is limited to a present Example and is not interpreted.

  In this example, an example is shown in which a luciferase gene is introduced into cells of the anterior tibial muscle of a mouse using the laser irradiation type foreign substance introduction device according to the present invention.

The laser irradiation type foreign substance introduction device used in this example is a colorless and transparent sapphire plate (IR system, thickness 1.5 mm, diameter 27 mm) as a light transmission body, and a shock wave generation layer. And black pigment (carbon black or aniline black, thickness 2 mm) and a colorless transparent sapphire plate (Ir System, Inc., thickness 1.0 mm, diameter 27 mm) as a shock wave transmitter, 1 is adopted. In this example, an Nd: YAG laser fundamental wave (wavelength 1064 nm, pulse width 10 nanoseconds, repetition frequency 10 Hz, irradiation area 0.5 cm ² , laser fluence 3 J / cm ² ) was used as the pulse laser light.

  First, the shin part of an anesthetized mouse (BALB / c, 8-10 weeks old, female, body weight about 20 g, Hokudo Co., Ltd.) was shaved, and TE solution (1 μg / μl) of luciferase gene expression plasmid (CAG promoter) was shaved. ) Was injected into the anterior tibial muscle (depth 1-2 mm). Next, after applying silicon grease to the shaved region of the front shin part, the laser irradiation type foreign substance introduction device was brought into close contact with the skin as shown in FIG. Finally, the device was irradiated with the pulse laser beam 200 to 500 times to generate a shock wave, and a luciferase gene expression plasmid was introduced into the cells of the anterior tibial muscle immediately below. After irradiation, the skin tissue of the anterior tibia was observed, but no burn symptoms were observed.

  Three days after the introduction of the plasmid, the introduction efficiency of the luciferase gene was examined. After the mice were sacrificed under anesthesia, the anterior tibial muscle was removed. The extracted anterior tibial muscle was homogenized in phosphate buffered saline containing 1% NP-40 three times its weight, and centrifuged to obtain a tissue extract. The luciferase activity contained in the tissue extract was measured using a Pickagene (Toyo Ink) and a luminometer (TD-20 / 20, Turner Design). The concentration of protein contained in the tissue extract was measured using a protein concentration measurement kit (Bio-Rad). The activity value of luciferase per unit protein amount was determined and used as the relative activity value of luciferase.

Table 1 is a table showing the relationship between the number of times of irradiation with pulsed laser light and the relative activity value of luciferase in this example. From this table, it can be seen that the luciferase gene introduction efficiency increases as the number of irradiations is increased.

  As a comparative example, an electroporation method, which is a typical conventional method, was used to introduce the same gene into cells of the same tissue with a treatment time of 50 seconds (a time corresponding to 500 irradiations in this example). As a result, the relative activity value by electroporation was 75. That is, the gene introduction efficiency using the device of the present invention was 133% with respect to the introduction efficiency of the electroporation method for the same processing time.

  As described above, according to the present invention, a gene can be safely introduced into cells in a living tissue with an introduction efficiency that exceeds that of the conventional method (electroporation method).

  The laser irradiation-type foreign substance introduction device according to the present invention can introduce a foreign substance such as DNA into cells in living tissue with higher introduction efficiency and safety than before. It is useful for development and functional analysis of genes.

The figure which shows the structure of the laser irradiation type foreign material introduction device which concerns on one embodiment of this invention The figure for demonstrating the foreign substance introduction | transduction method using the laser induced stress wave concerning one embodiment of this invention Diagram for explaining a conventional method for introducing foreign substances using laser-induced stress waves

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Polyethylene terephthalate 11 Black rubber disc 12 Laser beam 13 Laser induced plasma 14 Shock wave 15 Skin tissue 100 Laser irradiation type foreign substance introduction device 110 Light transmitting body 120 Shock wave generating layer 130 Shock wave transmitting body 140 Lens 150 Laser light 160 Biological tissue 170 cells 180 foreign substances

Claims (10)

A laser irradiation type foreign substance introduction device that introduces the foreign substance into the cells by giving a shock wave induced by laser light to cells in the living tissue into which the foreign substance is introduced in the vicinity,
A light transmitting body through which laser light emitted from the outside passes,
A shock wave generating layer that generates a shock wave by absorbing the laser light transmitted through the light transmitting body;
A shock wave transmitting body for transmitting a shock wave generated in the shock wave generating layer to the living tissue;
A laser irradiation type foreign substance introduction device.   The laser irradiation type foreign substance introduction device according to claim 1, wherein the light transmitting body is a sapphire plate.   The laser irradiation type foreign substance introduction device according to claim 1, wherein the shock wave transmitting body is a sapphire plate.   The laser irradiation type foreign substance introducing device according to claim 1, wherein the shock wave generating layer is a black pigment or a black rubber plate.   The laser irradiation type foreign substance introduction device according to claim 1, wherein the laser light is pulsed laser light.   The laser irradiation type foreign substance introduction device according to claim 5, wherein the pulse laser beam is a nanosecond pulse laser beam.   The laser irradiation type foreign substance introduction device according to claim 5, wherein the number of times of irradiation with the pulsed laser light is 10 to 1000 times. The laser irradiation type foreign substance introduction device according to claim 5, wherein a laser fluence of the pulse laser beam on an irradiation surface to the shock wave generation layer is ² to 10 J / cm ² .   The laser irradiation type foreign substance introduction device according to claim 5, wherein the pulsed laser light has a wavelength of 700 to 1500 nm.   The laser irradiation type foreign substance introduction device according to claim 1, wherein the foreign substance is any one of DNA, RNA, polypeptide, protein, lipid, and saccharide.

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