WO2024108566A1 - Transducteur à ultrasons, système de transfection in-vivo et leur utilisation - Google Patents

Transducteur à ultrasons, système de transfection in-vivo et leur utilisation Download PDF

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Publication number
WO2024108566A1
WO2024108566A1 PCT/CN2022/134406 CN2022134406W WO2024108566A1 WO 2024108566 A1 WO2024108566 A1 WO 2024108566A1 CN 2022134406 W CN2022134406 W CN 2022134406W WO 2024108566 A1 WO2024108566 A1 WO 2024108566A1
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WIPO (PCT)
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ultrasonic transducer
vivo transfection
photoresist
ultrasonic
transfection
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PCT/CN2022/134406
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English (en)
Chinese (zh)
Inventor
孟龙
刘秀芳
李鹏奇
郑海荣
牛丽丽
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中国科学院深圳先进技术研究院
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Priority to PCT/CN2022/134406 priority Critical patent/WO2024108566A1/fr
Publication of WO2024108566A1 publication Critical patent/WO2024108566A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N7/00—Ultrasound therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves

Definitions

  • the present application belongs to the field of bioengineering technology, and specifically relates to an ultrasonic transducer, an in vivo transfection system and applications thereof.
  • transfection The process of cells acquiring new phenotypes by introducing exogenous nucleic acid molecules under certain conditions is called transfection.
  • nucleic acids are macromolecules and can only enter cells through endocytosis, resulting in extremely low efficiency of nucleic acid uptake into cells. Therefore, it is necessary to use chemical, physical and biological methods to efficiently introduce target nucleic acid molecules into cells to achieve the function of target genes.
  • Transfection mainly includes ex vivo transfection and in vivo transfection. In vivo transfection is the process of directly injecting target exogenous genes into the living body and then achieving the function of target genes through chemical, physical and viral methods. Compared with ex vivo transfection, in vivo transfection is a direct and effective means to achieve gene and immunotherapy.
  • the neural network structure can be regulated by light or sound, which can provide a powerful tool for brain science research.
  • Gene therapy is also widely used in the treatment of brain gliomas, hereditary diseases, brain neurological diseases and other cancers. In vivo transfection also provides a new idea for disease treatment.
  • the most commonly used in vivo transfection techniques are viral transfection, cationic polymer transfection, electrofection, photofection, acoustic transfection, microinjection and gene gun.
  • the existing in vivo transfection technology has problems such as high cost, long time consumption, complex operation, high cytotoxicity and low broad spectrum, resulting in the inability of gene therapy to meet clinical needs.
  • transducers are mostly used to generate Gaussian beams to achieve fixed-point focusing.
  • the purpose of this application is to overcome the problems of high cost, long time consumption, complex operation, high cytotoxicity and low broad spectrum in the prior art.
  • an ultrasonic transducer for in vivo transfection which is prepared by the following method: spin coating photoresist on the upper surface of a piezoelectric substrate to form a photoresist layer; covering a mask plate on the photoresist layer for exposure, and after the light-transmitting part is cured, using a developer to dissolve the photoresist of the non-light-transmitting part; coating the piezoelectric substrate after photolithography; washing away the electrodes on the photoresist after coating; cutting the piezoelectric substrate into independent piezoelectric interdigital transducer units to obtain an ultrasonic transducer.
  • the technical solution adopted in the embodiment of the present application also includes: the above-mentioned piezoelectric substrate can be made of lithium niobate material or other piezoelectric materials or flexible materials.
  • the technical solution adopted in the embodiment of the present application also includes: in the above-mentioned ultrasonic transducer preparation method, the piezoelectric substrate after photolithography is placed in the chamber of the vacuum coating machine, and the equipment is turned on to perform sputtering coating of the target material.
  • the target material is preferably a nickel-chromium alloy target material, a gold target material, and silver, nickel and other materials can also be used.
  • the technical solution adopted in the embodiment of the present application also includes: in the above-mentioned ultrasonic transducer preparation method, acetone and an ultrasonic cleaning machine are used to wash away the electrodes on the photoresist after coating, and anhydrous ethanol is used to clean the residue and blow dry.
  • an in vivo transfection system comprising: a signal generator, a power amplifier, a fixing device and the above-mentioned ultrasonic transducer;
  • the signal generator is used to provide a sinusoidal pulse wave signal
  • the power amplifier is used to amplify the energy of the signal generated by the signal generator
  • the ultrasonic transducer is used to receive the energy-amplified signal and stimulate the experimental subject;
  • the fixing device is used to fix the experimental subject.
  • the experimental subjects may be living mice, rats, monkeys, rabbits, pigs, etc.
  • the technical solution adopted in the embodiment of the present application also includes: the above-mentioned in vivo transfection system also includes a gas anesthesia device; the gas anesthesia device is used to anesthetize the experimental subject.
  • the technical solution adopted in the embodiment of the present application also includes: the above-mentioned in vivo transfection system also includes a three-way device; the three-way device is used to extract blood from the experimental subject, and then return it to the experimental subject after ultrasonic treatment.
  • the present application also provides an in vivo transfection method, comprising the following steps:
  • the ultrasonic transducer receives the energy-amplified signal and stimulates the experimental subject in real time to complete in vivo transfection.
  • the ultrasonic transducer and in vivo transfection system provided in this application can be used for in vivo manipulation experiments, ultrasonic neural regulation, intracranial neuronal transfection, research on the specific mechanisms of gene disease treatment and immunotherapy, in vitro transfection of primary cells (T cells, macrophages), neuronal cells and difficult-to-transfect cell lines, and in vitro precise manipulation of particles and cell-related experiments.
  • the ultrasonic transducer provided in the present application optimizes the design of a new mode beam of Rayleigh wave conversion into body wave to suppress waveform distortion caused by complex media interfering with the phase and amplitude of the transmitted beam, overcomes the interference of diffraction and scattering effects on sound wave propagation, and achieves high-precision focusing in inhomogeneous media.
  • the in vivo transfection system provided by the present application has the advantages of low cost, high precision, easy operation, high safety and high broad spectrum.
  • In vivo transfection can be achieved without the introduction of microbubbles, avoiding the damage caused by the violent cavitation effect of microbubbles to normal tissues, and achieving accurate, efficient and safe in vivo transfection.
  • the system efficiently delivers nucleic acid macromolecule drugs into the brain through a new mode of acoustic wave conversion mechanism, avoiding the harmful inflammatory response caused by the opening of the blood-brain barrier, and achieving efficient, accurate and safe intracranial in vivo transfection, providing a powerful tool for the study of brain science.
  • the system can also achieve low-cost, easy-to-operate, accurate, efficient and safe CAR-T in vivo transfection, which can solve the high cost problem of in vitro culture of CAR-T cells and accelerate the clinical application of CAR-T immunotherapy.
  • the in vivo transfection system shows special advantages and huge application prospects in the field of genetic disease treatment due to its advantages of accuracy, safety, low cost and easy operation.
  • the in vivo transfection system can achieve perfect fit to any position of the living body through a flexible patch, it has a good broad spectrum.
  • FIG. 1 is a flowchart of manufacturing an ultrasonic transducer provided in an embodiment of the present application.
  • FIG. 2 is a schematic diagram of an ultrasonic transducer provided in an embodiment of the present application.
  • Figure 3 is the Z-direction sound field action range of the ultrasonic transducer provided in an embodiment of the present application; (a) the concentric annular array interdigitated geometric model established by finite element numerical simulation; (b-d) the sound pressure distribution on each xy, yz, and zx section.
  • FIG. 4 is a diagram showing the Z-direction sound field range of the ultrasonic transducer characterized by the laser Doppler vibrometer.
  • Embodiment 1 is a diagrammatic representation of Embodiment 1:
  • transducers are mostly used to generate Gaussian beams to achieve fixed-point focusing.
  • the reflection, refraction, scattering and absorption of sound waves by biological tissue, a non-uniform and complex medium disrupts the synthesis of the focused beam.
  • the new mode beam of Rayleigh wave conversion body wave is optimized to suppress the waveform distortion caused by the interference of complex media on the phase and amplitude of the transmitted beam, and achieve high-precision focusing in non-uniform media.
  • the ultrasonic transducer used in this embodiment is a type of Rayleigh wave transducer, which is mainly made by plating interdigital electrodes on a piezoelectric substrate.
  • a high-frequency Rayleigh wave out-of-plane chip is constructed by using the high vibration velocity on the elastic piezoelectric solid substrate surface.
  • the wavefront propagates at the fluid-solid/solid-solid interface and undergoes abnormal refraction, forming Rayleigh angle out-of-plane propagation, and relying on the Rayleigh wave leakage mode to form a body wave ultrasonic sound field in the homogeneous medium.
  • Gluing On the surface of the completely cleaned piezoelectric single crystal, the positive photoresist AZ5214 was first spin-coated at 500 rpm for 15 seconds, and then at 3000 rpm for 30 seconds. The piezoelectric substrate was placed on a 120°C heating plate and baked for 3 minutes. The thickness of the photoresist was tested using a step profiler, and the thickness of the photoresist was 5 ⁇ m, as shown in Figure 1(a).
  • the traditional solution is limited by the thickness vibration mode of the piezoelectric substrate, and the transducer device is in a low-frequency application scenario and cannot meet the requirements of high spatial resolution.
  • This embodiment realizes high-frequency ultrasonic modulation of the sound field by rearranging the electrodes and utilizing the mode conversion at the coupling interface. Since the single electrode unit can work on a flexible patch, this provides a new solution for real-time monitoring of wearable transducers.
  • the high light transmittance of the substrate makes it possible to implant optical fibers, electrodes, etc., and to build an in-situ stimulation real-time detection system, which is convenient for the screening of ultrasonic parameters and real-time efficacy evaluation.
  • Ultrasonic energy directly determines the in vivo transfection effect, so the screening of ultrasonic parameters is crucial.
  • Ultrasonic parameters mainly include: fundamental frequency, pulse repetition frequency, duty cycle, pulse length, pulse duration, stimulation interval, sound intensity, sound pressure and total stimulation duration.
  • sound pressure mostly refers to negative sound pressure
  • sound intensity mostly refers to the spatial peak pulse average intensity and spatial peak time average intensity.
  • a network analyzer is used to measure the insertion loss of each frequency of the transducer by frequency sweeping, and the frequency point with low insertion loss is selected as the driving frequency of the transducer to ensure sufficient output of sound field energy.
  • the ultrasonic sound field generated by the ultrasonic transducer In order to achieve the range of the sound field and the characterization of the sound wave energy, it is necessary to measure the ultrasonic sound field generated by the ultrasonic transducer.
  • the slow-speed surface distribution of the anisotropic piezoelectric substrate is measured, and the surface sound field information of the piezoelectric substrate is measured using a laser Doppler vibrometer.
  • the laser beam is emitted to the surface of the piezoelectric substrate, and the reflected laser beam produces a Doppler frequency shift due to the movement of the surface, from which the vibration amplitude and frequency of the piezoelectric substrate surface are extracted to obtain the sound pressure information and distribution.
  • a needle hydrophone is used to measure the full-space sound field, and based on the piezoelectric effect of the piezoelectric film of the needle hydrophone, the full-space sound field and sound pressure information are obtained.
  • the results are shown in Figures 3-4.
  • the center frequency of the Rayleigh wave transducer is 19.65MHz; the focused sound field with a focal length of about 1.2mm and a focal spot of about 140 ⁇ m in the Z direction can be used for in vivo precision transfection research.
  • a high-frequency Rayleigh wave out-of-plane chip is constructed using the high vibration velocity on the elastic piezoelectric solid substrate.
  • the wavefront undergoes abnormal refraction at the fluid-solid/solid-solid interface, forming Rayleigh angle out-of-plane propagation, and relying on the Rayleigh wave leakage mode to form a body wave ultrasonic sound field in the homogeneous medium.
  • the influence of the arrangement of metal electrodes on the anisotropic substrate on the performance of the modulated sound field is studied, and the relationship between the thickness of the isotropic propagation medium and the target ultrasonic sound field is further explored.
  • AML Acute myeloid leukemia
  • AML cell line MV4-11 (carrying Luciferase gene) was selected, 3 ⁇ 106 cells were collected and dissolved in 500 ⁇ L PBS solution, and after thorough mixing, they were injected into Prkdc gene and Il2rg gene knockout immunodeficient (NSG) mice irradiated with 100-200cGy for 6h by tail vein injection. 4-6 weeks after injection, the expression of tumor cells was observed by fluorescence using small animal in vivo bioluminescence imaging technology to evaluate the proliferation and expansion of tumor cells and determine whether the model was successfully established.
  • the in vivo transfection system comprises: a signal generator, a power amplifier, the ultrasonic transducer, a fixing device, an air anesthesia device, a three-way device, and the mouse model.
  • the signal generator provides a sinusoidal pulse wave signal for the ultrasonic transducer;
  • the power amplifier amplifies the energy of the signal generated by the signal generator;
  • the ultrasonic transducer realizes high-energy, precise focused stimulation of the experimental body;
  • the fixing device can stably expose the stimulation position of the mouse;
  • the gas anesthesia device is combined with the fixing device to ensure that the stimulation position of the mouse continuously and stably receives ultrasonic stimulation;
  • the three-way device realizes a function similar to that of a kidney dialysis device, extracts blood from the model mouse, and after ultrasonic treatment, returns it to the mouse.
  • the fluorescently labeled CAR-T plasmid was injected into the mouse model through the tail vein, and the mouse was fixed in the fixing device of the in vivo transfection system to perform CAR-T in vivo transfection on the model mouse.
  • the ultrasonic transducer provided in this application optimizes the design of the new mode beam of Rayleigh wave conversion body wave to suppress the waveform distortion caused by the interference of complex media on the phase and amplitude of the transmission beam, overcome the interference of diffraction and scattering effects on the propagation of sound waves, and achieve high-precision focusing in non-uniform media.
  • the in vivo transfection system provided in this application uses the characteristics of high energy focusing to achieve precise control, thereby achieving precise transfection at a specific position. Low cost, easy operation, and can achieve accurate, efficient and safe CAR-T in vivo transfection.
  • the system not only uses the standard MEMS method to prepare a flexible array transducer to achieve multi-target neuronal stimulation, but also provides a powerful tool for brain research, and shows special advantages and huge application prospects in the field of genetic disease treatment. At the same time, it can also solve the high cost problem of in vitro culture of CAR-T cells and accelerate the clinical application of CAR-T immunotherapy.
  • the system is not limited to the location and type of living body, and can achieve in vitro transfection of primary cells (T cells, neurons, etc.) and difficult-to-transfect cell lines; it has a high spectrum and can achieve any position in the body, including intracranial transfection. Provide new treatment ideas for brain glioma, genetic diseases, brain neurological diseases, etc.

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Abstract

La présente invention concerne un transducteur à ultrasons pour la transfection in vivo. Le transducteur à ultrasons est préparé selon le procédé suivant : revêtement par centrifugation d'une résine photosensible sur la surface supérieure d'une matrice piézoélectrique pour constituer une couche de résine photosensible ; recouvrement de la couche de résine photosensible par une plaque de masquage et exposition, et après durcissement de la résine photosensible dans la partie masquée, dissolution de la résine photosensible dans la partie non masquée à l'aide d'une solution de développement ; après la photolithographie, dépôt d'un film sur la matrice piézoélectrique ; lavage pour éliminer les électrodes sur la résine photosensible recouverte du film ; et découpage de la matrice piézoélectrique en unités indépendantes de transducteurs interdigités piézoélectriques pour obtenir les transducteurs à ultrasons. Le transducteur à ultrasons peut supprimer la distorsion de la forme d'onde causée par l'interférence de phase et d'amplitude du faisceau de transmission dans un milieu complexe, ce qui permet d'obtenir une focalisation de haute précision dans un milieu hétérogène. Le système de transfection in vivo construit sur cette base présente les avantages d'une efficacité de coût, d'une précision élevée, d'une facilité de fonctionnement, d'une sécurité élevée et d'un large spectre. La transfection in vivo peut être obtenue sans introduire de microbulles, ce qui permet d'éviter l'endommagement de tissus normaux provoqués par l'effet de cavitation sévère des microbulles, et d'obtenir une transfection in vivo précise, efficace et sûre.
PCT/CN2022/134406 2022-11-25 2022-11-25 Transducteur à ultrasons, système de transfection in-vivo et leur utilisation WO2024108566A1 (fr)

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CN110548233A (zh) * 2019-09-30 2019-12-10 上海交通大学 一种用于神经调控的便携式双通道经颅超声刺激装置
CN114644982A (zh) * 2022-04-25 2022-06-21 深圳高性能医疗器械国家研究院有限公司 基因转染方法和基因转染装置
CN114717267A (zh) * 2022-04-15 2022-07-08 深圳高性能医疗器械国家研究院有限公司 基因转染方法及系统

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001053569A (ja) * 1999-08-16 2001-02-23 Hitachi Denshi Ltd 弾性表面波素子の製造方法
CN101555450A (zh) * 2008-04-08 2009-10-14 清华大学 一种实现对单细胞或小组织内部的注射和吸引的超声装置
US20100156244A1 (en) * 2008-09-18 2010-06-24 Marc Lukacs Methods for manufacturing ultrasound transducers and other components
KR20130097552A (ko) * 2012-02-24 2013-09-03 경북대학교 산학협력단 다층 능동재료형 초음파 트랜스듀서 및 그의 제조 방법
CN104548390A (zh) * 2014-12-26 2015-04-29 中国科学院深圳先进技术研究院 一种超声深部脑刺激方法及系统
CN108039873A (zh) * 2017-11-30 2018-05-15 深圳华远微电科技有限公司 一种芯片级声表面波滤波器制作方法
CN108477675A (zh) * 2018-02-28 2018-09-04 云南中烟工业有限责任公司 一种电子烟雾化芯片的制备方法
CN108511600A (zh) * 2018-02-28 2018-09-07 云南中烟工业有限责任公司 一种声致雾化芯片的制备方法
CN110548233A (zh) * 2019-09-30 2019-12-10 上海交通大学 一种用于神经调控的便携式双通道经颅超声刺激装置
CN114717267A (zh) * 2022-04-15 2022-07-08 深圳高性能医疗器械国家研究院有限公司 基因转染方法及系统
CN114644982A (zh) * 2022-04-25 2022-06-21 深圳高性能医疗器械国家研究院有限公司 基因转染方法和基因转染装置

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