JP4477922B2 - Sonic micro-drilling device - Google Patents

Description

The present invention relates to a sonic fine hole punching device that makes fine holes in the surface of cells / tissues in water by sonic energy.

  As a conventional technique for forming a large number of fine holes having a diameter of a micro unit or a nano unit on the surface of a film in water or an individual, there are techniques such as a laser method, an electroporation method, and a micro needle method.

Sonic energy and ultrasonic energy have been utilized for cleaning and crushing as industrial applications. In addition, ultrasonic energy has been widely used in the medical field for diagnosis of heart diseases, abdominal diseases, head and neck diseases, and the like. On the other hand, it is also used for therapeutic purposes, in which gallstones and kidney stones in the body are crushed by strong sound waves (shock waves) and the sonic energy is converted into thermal energy to burn the cancer locally. Research is also underway to apply ultrasonic energy to help drug penetration into tissues. (See Patent Documents 1 and 2).
US Pat. No. 6,676,963 US Pat. No. 6,528,039

  It is difficult to make a large number of fine holes (1 to 10 nanometers in diameter) (1 to 1 million per millimeter square) on the surface of a thin film or an individual in water. Lasers are unsuitable for making fine holes because they are absorbed and scattered in water. In addition, it is difficult to make a large number of fine holes stably and efficiently with a microneedle or an electroporation method. There is also a drilling technique that utilizes an intermediate phenomenon between the destructive action and non-destructive action of sonic energy. Ultrasonic energy creates fine bubbles (0.1 mm to 0.1 micron in diameter) in water, ruptures them with sound waves, and instantly creates a hole in the surface of a membrane or solid using a jet stream Attempts have been made. However, it is extremely difficult to control the generation and destruction of bubbles. The main reason is that the target sound field has a very complicated and non-linear nature, so that it is almost impossible with a general-purpose sound wave oscillating element and a driving power source that generate a single frequency, sound pressure, and waveform. Moreover, in order to create an appropriate sound field, specialized knowledge and advanced technology are required, it is limited to a limited state, and there are very few uses. However, researchers and engineers who are not specialized in acoustics often face the need to drill fine holes in a variety of situations. If a simple hole can be made, this phenomenon can be applied to a wide range of general purposes.

  Further, when the sound wave transmission vibration part is brought close to or directly in contact with a target to be drilled, the mechanical destructive action is enhanced, and the target object is often destroyed before a fine hole is drilled. This is a major obstacle in creating a stable sound field and optimum conditions for drilling holes. There is currently no device that can control the breaking action and make a minute hole in the immediate vicinity of the surface of the vibrating part (around 2 mm).

  Therefore, the present invention provides a sonic micro-drilling device that can make a fine hole on the surface of an individual without using a large-scale device and can suppress the destructive action in the immediate vicinity of the hole. Is.

  The sonic fine hole punching device of the present invention is divided into a sound wave oscillating portion and a drive power source portion, and fine holes can be made accurately and stably only when they are optimized for certain conditions. The size, number, and shape of the oscillating portion of the sound wave are set and created in advance, and are selected according to the purpose.

  Further, the frequency, amplitude, waveform, and pulse switching of signal transmission of the drive power supply device that supplies electric energy are set in advance by a program. On the other hand, by feeding back the electrical impedance, temperature, resonance information, etc. from the sensor installed in the sound wave oscillation part to the CPU of the main body of the device, the oscillation drive signal is adjusted again, and the hole is most stably opened. Control to the state. The electric elements of the electric signal to be adjusted and controlled include frequency (1 Hz to 20 MHz), voltage (0.1 V to 200 V), waveform, burst wave time, burst wave repetition frequency (0.01 Hz to 100 Hz), phase (from 0 360 degrees), duty ratio (0 to 100%), pulse wave on / off frequency (0.01 Hz to 100 Hz), and the like. Electrical power from a minimum of 1 mW to a maximum of 400 W is required.

  As shown in FIG. 1 (a), which is different from simple waveforms such as rectangular wave, sine wave, and sawtooth wave, which have been widely used as the transmission signal waveform, it is more efficient than before by using an asymmetric waveform. I can make a hole. The signal waveform needs to have a sharper peak waveform with an asymmetrical plus-minus voltage difference.

  On the other hand, a stable drilling effect can be obtained by strict switching of on / off in the burst wave or pulse wave of the sound wave oscillation. Generally, it is possible to temporarily stop the oscillation of sound waves by setting the drive electrical signal to zero voltage, but completely eliminate the natural vibration of the oscillating element immediately after the vibration stops and the residual sound of the surrounding sound field. Therefore, a silent state can be positively created by applying a voltage having a reverse phase of 180 degrees immediately after the end of the drive signal. By creating such a strict silent state frequently, it is possible to create a state in which fine holes are efficiently and stably opened. A similar phenomenon shown in FIG. 1B appears when a sound wave is generated with a signal having a randomly changing frequency or when the sound wave oscillating unit is driven with so-called white noise. The resonance frequency varies depending on the size, shape, and material of the sound wave oscillating part, but when a random frequency is driven for each block, sound wave energy is generated when it exactly matches the resonance frequency. Therefore, the vibration stops rapidly and a silent state occurs. By repeating this, sonic energy can be used effectively. The frequency change of random or white noise is kept within plus or minus 10% of the center frequency, and is generated within the range according to the random number table. The rate of change is 1 Hz to 300 Hz. The present invention has the basic feature that this principle is incorporated in a program circuit of a drive power supply portion and sonic energy is efficiently oscillated.

  In FIG. 2, regarding the protection of the sound wave oscillating element 1, a PZT vibrator or the like is formed by a protective film 2 made of polyethylene, nylon, urethane, silicon, etc. that is electrically insulating and has good sound wave transmission. By covering the periphery of the vibration part 3, it is possible to avoid unnecessary destructive action and to efficiently make fine holes in the target object. The thickness of the protective film is about 10 microns to 1 cm and does not necessarily have to be captured in the form of a sonic vibration element.

  Further, as shown in FIG. 3, ultrapure water distilled water is used as a fluid 4 that is electrically insulative and has good sound wave transmission around the sound wave oscillating element 1 connected to the sound wave element driving electrode 1a. The same effect can be obtained by encapsulating hydrogel, liquid paraffin, oil, lipid and the like together with the sound wave oscillating element 1 in a thin film 5 such as polyethylene, nylon, urethane, or silicon. The thickness of the coating 5 may be about 10 microns to 1 cm. In this case, the distance between the vibration surface and the coating film needs to be the same as or longer than the amplitude of the vibration surface. In addition, it is necessary to adjust the viscosity so that bubbles do not occur in the fluid encapsulated in the coating. When the acoustic vibration element is a flat surface, the central portion has the largest vibration amplitude, and therefore it is necessary to coat the vibration surface of the portion thickly with a solid or fluid. In addition, 6 is an impedance electrode for measuring the resistance of the fluid 4, and 7 is a temperature sensor.

  According to the device of the present invention, a fine hole can be formed on the surface of an individual without using a large-scale device, and the destructive action around the hole can be suppressed.

  Attempts have been made to inject various substances such as genes, drugs and chemical substances into cells and tissues in research in the medical field and biological research field. For this purpose, it is necessary to make fine holes in the surface of the cell / tissue.

  As shown in FIG. 4, the sound wave oscillating element 1 of the device of the present invention is inserted through a protective film 2 into a cell culture vessel 8 containing a large number of cancer cell culture strains, and a substance to be injected into cancer cells is added and a drive power source When activated, sonic energy is irradiated to the cancer cells, and many fine holes are made. From this hole, genes, drugs, and chemicals flow into the cancer cells. Since most living cells exist only in an environment close to an aqueous solution, it is meaningful to carry out the present invention in water. In the conventional technique, the cells may be destroyed by the sound wave energy. However, since the periphery of the sound wave oscillating portion of the present invention is coated, damage to the cells is suppressed to the minimum. In addition, by using driving signals such as random frequency, white noise, and antiphase wavelength, fine holes can be evenly formed in each cell. As another form of use of the sound wave oscillating element, if the sound wave vibration unit is incorporated in the bottom portion of the cell culture vessel 6 in advance, the trouble of newly inserting the sound wave oscillation device into the water can be saved. It becomes possible to irradiate a large number of cells with sound waves simultaneously. In addition, by reducing the size of the acoustic vibration unit, it is possible to efficiently make holes in cells even with a very small amount of cell culture solution. In order to drive a large number of sound wave oscillating elements 1 at the same time, it is also possible to connect the electric supplies of the respective elements 1 in parallel to the sound wave element driving electrodes 1a.

  In FIG. 5, 11 is a frequency display, 12 is a pulse frequency and duty ratio display, 13 is an output display, 14 is a timer time display, 15 is a temperature display, 16 is a frequency adjustment knob, 17 is a pulse adjustment knob, and 18 is a duty ratio. An adjustment knob, 19 is an output adjustment knob, 20 is a timer setting knob, and 21 is a white / random frequency switching knob. The driving device displays the measured temperature, measured voltage, measured electrical energy (watt) consumption, measured drive frequency (frequency counter), duty ratio (%), and burst frequency (Hz) of the sound wave oscillation part. In addition, the driving power supply can be turned on and off by an automatic timer (0.1 to 3600 seconds). A changeover switch for optimizing the drive signal is also installed based on each information fed back from the sonic vibration site. There is also provided a changeover switch for an effective use mode of sound wave energy using drive signals such as the above-mentioned random frequency, white noise, and antiphase wavelength.

  In an animal as well as a cultured cell, it is possible to make a fine hole by irradiating a target site using a sound wave oscillator. It is also possible to arrange several sound wave oscillating parts in a straight line and insert them into the digestive tract or blood vessel to open many fine holes in the surrounding cells or living tissue. Central nervous system drugs, peripheral nervous system drugs, sensory organ drugs, cardiovascular drugs, respiratory organ drugs, gastrointestinal drugs, hormone drugs, urogenital drugs, skin drugs, through numerous fine holes Thrombolytic agent, antitumor agent, antiallergic agent, antibiotic, metabolic drug, gene therapy agent, diagnostic contrast agent, staining agent, photosensitizer, ultrasound agent, vasodilator, neovascular inhibitor, Vitamins, antioxidants, anti-inflammatory agents, etc. can be administered efficiently. It can also be applied to experiments such as cloning techniques for animal eggs, chicken embryos, mouse embryos, cow embryos and the like. Even when a fine hole is made in a cell wall of a plant, it can be used for breeding by using the apparatus of the present invention. As an industrial application, it can be used for making many holes in the surface of a film or material that cannot be dried.

  In another embodiment, a microcapsule or nanometer size lipid, a polymer particle or a capsule containing a gas may be used together when using the device to more efficiently perforate. In general, the ultra-contrast and MRI contrast agents that are currently used and the nano-percentles (nano-sized microparticles) of diagnostic and therapeutic drugs that have the property of accumulating in specific tissues can be mixed in a liquid and used in combination. Many holes can be made. It is possible to adjust the size of the holes and the number per hole area.

[Reference Example 1]
A 1 cm square aluminum foil is immersed in water, and a random frequency of about 2 megahertz (± 0.1 megahertz), 10 watt square centimeters, a pulse frequency of 30 hertz, is irradiated from a distance of 1 mm on the surface by the sound wave oscillator of the present invention. In a time of 60 seconds, 10,000 fine holes with a size of 10 microns could be made on average of 1 square mm.

The apparatus of the present invention is used when a gene is introduced into a chicken embryo limb bud (stages 20 to 21). First, a chicken egg is generated in a 38.5 ° C. incubator keeping the humidity until the target stage is reached. A hole is made in the blunt end of the eggshell, and about 4-5 ml is extracted with a syringe with an 18G needle. Cut the upper side of the eggshell with an ophthalmic scissors and open a round window. The gene {GFP (pEGFP), LacP (pEF-LacZ)} is directly injected into the limb bud under a microscope. The sound wave vibrator of the present invention is inserted close to the target location. The conditions for introducing a gene by opening a hole in the target site are ultrasonic energy of 2.0 W / cm ² , duty cycle of 20%, and irradiation time of 60 seconds. Run in random frequency mode. As a result, the expression vector was introduced into the limb bud, and 50 times the conventional electroporation was obtained. The expression of the GFP gene was transient, the gene was already expressed 3 hours after the introduction, reached the peak of expression almost 12 hours later, and almost no expression was observed by 48 hours LacZ Gene transfer was also confirmed in limb buds 12 hours after gene transfer. By using the same procedure, it was confirmed that an excessive finger could be induced when an expression vector (pCAGGS-cShh) for the Shh gene was used.

A cavity that reaches the pulp is formed from the root side of the freshly extracted bovine anterior tooth using a diamond diamond round bar # 440, and a plasmid (TIMP-pEGFP) in which TIMP promoter is bound to pEGFP-N3 (Clontech, Palo Alto) is used. Injected. The plasmid DNA (25 μg) was previously mixed with an ultrasound contrast agent Optison (Molecular Biosystems Inc. San Diego) 1: 3. The cavity was completely covered with Ultra / Ponic Conductive Gel solution (Nishimoto Sangyo Co. LTD), and a sound wave was irradiated at 1 MHz from the Gel surface using the device of the present invention. The ultrasonic intensity was 0.5 W / cm ² or 1 W / cm ² and the frequency was 1 MHz (irradiation time 15, 30, 60 seconds). Thereafter, the dental pulp was removed, and Trowell type organ culture was performed in DMEM containing 10% calf serum. After 24 hours, GFP fluorescence was observed with a stereofluorescence microscope.

As a result, the amount of GFP in the group irradiated with ultrasonic waves was significantly recognized, and it seems that many fine holes were formed in the tissue and the film on the surface. The effect of promoting the gene transfer efficiency was proved, and the transfer efficiency depended on the intensity of the ultrasonic wave and the irradiation time. In the case of the frequency of 1 MHz of the device of the present invention, the intensity is 0.5 W / cm ² , and the irradiation time of 30 seconds in the white noise mode is the most diffusely distributed in the pulp tissue in the cavity and there is no great tissue damage. The gene transfer efficiency was considered to be the best.

The effect on vascular endothelial tissue was tested. 20 μg of TIMP-pEGFP plasmid is applied to the vascular endothelial tissue, and ultrasonic irradiation is performed from the sonic oscillation part of the device of the present invention at 0.5 W / cm ² , frequency 1 MHz, irradiation time 30 seconds, and the tissue is rapidly cultivated 1 day after organ culture. Frozen. A 20 μm frozen section was prepared, and the fluorescence development of intracellular GFP was observed with a confocal laser microscope (Carlz Ice, LSM410, argon laser, B 488 nm, 30 mW). In addition, observation of inflammation and necrosis was performed by HE staining.

  As a result, vascular tissue one day after organ introduction and vascular tissue was stained with HE and observed for morphology. No inflammation or necrosis was observed. In addition, when GFP fluorescence was analyzed by a computer image under a confocal laser microscope, it was found that the GFP gene was incorporated into the cells up to 200 μm below the surface layer by gene introduction.

  The device of the present invention was inserted into an artificial thrombus created in the blood vessel of the abdominal aorta of a rabbit, irradiated with 1 watt, 2 megahertz, and 30 seconds of sound waves and added with a thrombolytic agent. Thrombolysis was observed in a significantly shorter time than the group without. It seems to be the result of opening many fine holes on the surface of the thrombus.

It is a figure of the waveform used with this invention apparatus. It is a figure which shows one Example of this invention apparatus. It is a figure which shows another Example of this invention apparatus. It is a figure which shows another Example of this invention apparatus. It is a figure which shows an example of the drive device of this invention apparatus.

Explanation of symbols

1: Sound wave oscillating element 1a: Sound wave element driving electrode 2: Protective film 3: Vibrating part 4: Fluid 5: Film 6: Impedance electrode 7: Temperature sensor 8: Cell culture vessel 11: Frequency display 12: Pulse frequency and duty ratio Display 13: Output display 14: Timer time display 15: Temperature display 16: Frequency adjustment knob 17: Pulse adjustment knob 18: Duty ratio adjustment knob 19: Output adjustment knob 20: Timer setting knob 21: White / random frequency switching knob

Claims (2)

In a sonic micro-drilling device that punctures the surface of cells and tissues in water with sonic energy, the thickness of the oscillating element where the positive and negative sides of the oscillating drive signal waveform are asymmetric and sharp peaks are thick. A sonic micro-drilling device, which is covered with an electrically insulating and sound-transmitting film having a thickness of 10 microns or more. 2. A sonic micro-drilling device according to claim 1, wherein a fluid that is electrically insulative and has good sound wave transmission is enclosed in a film together with the sonic oscillation element around the sonic oscillation element .

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