WO2022021734A1 - 一种超声声场适形的基因转染装置 - Google Patents

一种超声声场适形的基因转染装置 Download PDF

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Publication number
WO2022021734A1
WO2022021734A1 PCT/CN2020/135338 CN2020135338W WO2022021734A1 WO 2022021734 A1 WO2022021734 A1 WO 2022021734A1 CN 2020135338 W CN2020135338 W CN 2020135338W WO 2022021734 A1 WO2022021734 A1 WO 2022021734A1
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Prior art keywords
ultrasonic
sound field
conformer
gene transfection
ultrasonic sound
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PCT/CN2020/135338
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English (en)
French (fr)
Inventor
严飞
蒋天安
谢丽婷
邹俊杰
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中国科学院深圳先进技术研究院
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Publication of WO2022021734A1 publication Critical patent/WO2022021734A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli

Definitions

  • the present application relates to the field of scientific research devices used in biomedical experiments, in particular to a gene transfection device conforming to an ultrasonic sound field.
  • Gene therapy is the introduction of normal genes or therapeutic genetic material into target cells in a specific way to correct gene defects or play a therapeutic role, so as to achieve the purpose of treating diseases.
  • the three elements of gene therapy are target gene, gene transfection and target cells.
  • gene transfection technology is the key to determine the effect of gene therapy.
  • gene delivery systems that are non-invasive, highly targeted, highly controllable, and can be effectively expressed are particularly important in clinical practice.
  • how to safely and efficiently deliver genes to the target site and stably and persistently express them in tissue cells is a major problem and challenge for gene therapy.
  • UTMD ultrasound targeted microbubble destruction
  • the principle is to use gas-containing ultrasonic microbubbles to continuously vibrate, expand, shrink, and rupture under the ultrasonic irradiation of a certain sound intensity and mechanical index, resulting in an instantaneous cavitation effect that increases the permeability of the cell membrane and causes it to break down.
  • Temporary, reversible sonopores are created in the cell membrane through which genes bound to microvesicles can enter the cell.
  • UTMD has broad application prospects.
  • the existing UTMD still has its application flaws.
  • the existing UTMD ultrasonic transfection apparatus usually uses culture vessels such as culture dishes, culture plates, etc. to carry the complexes of cells, genes and ultrasonic microbubbles, and performs ultrasonic irradiation through a focused ultrasonic probe.
  • the distribution of the sound field irradiated by the probe is not uniform, the sound field can only cover a part of the culture vessel, and the cells are usually scattered in various parts of the culture vessel, which leads to the low utilization rate of the irradiation energy of the ultrasonic probe, which in turn leads to low gene transfection efficiency. .
  • the present application provides a gene transfection device conformal to the ultrasonic sound field, which can realize the conformal coverage of the ultrasonic sound field, thereby maximizing the utilization of ultrasonic energy, thereby improving the gene transfection efficiency.
  • an ultrasonic sound field conformal gene transfection device comprising: an ultrasonic transducer for emitting ultrasonic waves; an ultrasonic sound field conformal device for accommodating a complex of genes, cells and ultrasonic responsive particles, The shape and size of the ultrasonic sound field conformer are determined according to the ultrasonic sound field; the focal length adapter is used to fix the ultrasonic sound field conformer in a preset position, and the preset position is based on the ultrasonic sound field location is determined.
  • the ultrasonic sound field conformer and the ultrasonic transducer are adapted to each other, and the two have a corresponding relationship.
  • the size and shape of the ultrasonic sound field conformer are determined according to the sound field of the ultrasonic waves emitted by the ultrasonic transducer, and the ultrasonic sound field conformer is further fixed at a preset position, which is located in the sound field and is based on the sound field.
  • the ultrasonic sound field position is determined, and the ultrasonic sound field conformer and the ultrasonic sound field conformally cover each other, so as to maximize the utilization of ultrasonic energy, thereby improving the gene transfection efficiency.
  • the target transmission characteristics and instantaneous reversible permeability can be improved.
  • Ultrasound irradiation itself has organ targeting, which can improve the local gene transfection efficiency of irradiation. Based on a certain power of ultrasound irradiation, the ultrasound-responsive particles will rupture and release the drugs or genes they carry. Ultrasound responds to particle rupture to produce cavitation effects, including sonoporous effects, microfluidics, etc., which instantly and reversibly increase the permeability of microvessels or cell membranes and barriers in vivo, which can promote gene penetration. Therefore, the gene transfection device provided in the present application further enables the gene transfection technology of cells to achieve efficient transfection and universality, and has a very broad application prospect.
  • the ultrasonic-responsive particles contained in the ultrasonic sound field conformer may be nano-particles or micro-particles, such as biological nanobubbles.
  • the ultrasound-responsive particles may be ultrasound microbubbles.
  • the ultrasonic microbubbles may be lipid ultrasonic microbubbles, such as phospholipid bubbles, PLGA microbubbles, and the like.
  • the cells accommodated by the ultrasonic sound field conformer can be any one of suspension cells, adherent cells, stem cells, or primary cells.
  • the shape of the ultrasonic sound field shaper is determined according to the shape of the focal spot of the sound field
  • the size of the ultrasonic sound field shaper is determined according to the size of the focal spot
  • the The preset position is determined according to the position of the focal spot.
  • the shape of the ultrasonic sound field conformer is the same as the shape of the focal spot.
  • the size of the ultrasonic sound field conformer is the same as the size of the focal spot.
  • the preset position is the center position of the focal spot.
  • one end of the focal length adapter is used for connecting with the ultrasonic sound field conformer, and the other end of the focal length adapter is used for connecting with the ultrasonic transducer.
  • the ultrasonic sound field conformer is located at the preset position.
  • the focal length adapter provided in the embodiment of the present application can play the role of focus positioning.
  • the ultrasonic sound field shaper can be automatically located in the The preset position, so there is no need to manually adjust the position of the ultrasonic sound field conformer, thereby simplifying the operation steps and ensuring the convenience and efficiency of gene transfection.
  • the ultrasonic sound field conformer and the focal length adapter form an integral structure through an integral molding process.
  • the ultrasonic sound field conformer and the focal length adapter are integrally formed by 3D printing technology.
  • it can be integrally formed from photosensitive resin, plastic, rubber, glass, metal, etc. through 3D printing technology.
  • the ultrasonic sound field conformer is fixedly connected inside the focal length adapter.
  • the focal length adapter includes a top wall, the ultrasonic sound field conformer is fixedly arranged on the inner wall surface of the top wall, a sample loading port is opened on the top wall, and the sample loading port is opened on the top wall.
  • the port communicates with the opening of the ultrasonic sound field conformer.
  • the top wall is provided with an exhaust hole.
  • a concave structure is formed in the middle of the top wall, the height of the inner surface of the concave structure is lower than the height of the inner surface of other parts of the top wall, and the sample loading port is opened in the inner surface. inside the concave structure.
  • the concave structure By setting the concave structure, on the one hand, it can facilitate feeding and prevent the compound from flowing into the external environment; , the air bubbles will be displaced to other parts of the top wall, so that the propagation of ultrasonic waves will not be affected by the air bubbles, avoiding the problem of energy attenuation, which is beneficial to improve the efficiency of gene transfection. At this time, even if the sample inlet is not provided, it can also play the role of removing air bubbles.
  • the focal length adapter further includes a peripheral wall, one end of the peripheral wall is connected to the top wall and arranged around the circumference of the top wall, and the other end of the peripheral wall is provided with a connector,
  • the focal length adapter is detachably connected to the ultrasonic transducer through the connector.
  • the focal length adapter is detachably connected with the ultrasonic transducer through the connector, so that flexible and simple connection between the two can be realized. At the same time, it is convenient to replace and use different ultrasonic transducers to meet the needs of different experiments or treatments.
  • the ultrasound transducer is a focused ultrasound probe.
  • the ultrasound-responsive particles are ultrasound microbubbles.
  • the ultrasonic transducers and the ultrasonic sound field conformers each include a plurality, and the plurality of ultrasonic transducers correspond to the plurality of the ultrasonic sound field conformers one-to-one.
  • the sound field of the ultrasonic waves emitted by the transducers varies.
  • the ultrasonic sound field conformer and the ultrasonic transducer are adapted to each other, and the two have a corresponding relationship.
  • the size and shape of the ultrasonic sound field conformer are determined according to the sound field of the ultrasonic waves emitted by the ultrasonic transducer, and the ultrasonic sound field conformer is further fixed at a preset position, which is located in the sound field and is based on the sound field.
  • the ultrasonic sound field position is determined, and the ultrasonic sound field conformer and the ultrasonic sound field conformally cover each other, so as to maximize the utilization of ultrasonic energy, thereby improving the gene transfection efficiency.
  • the target transmission characteristics and instantaneous reversible permeability can be improved.
  • Ultrasound irradiation itself has organ targeting, which can improve the local gene transfection efficiency of irradiation. Based on a certain power of ultrasound irradiation, the ultrasound-responsive particles will rupture and release the drugs or genes they carry. Ultrasound responds to particle rupture to produce cavitation effects, including sonoporous effects, microfluidics, etc., which instantly and reversibly increase the permeability of microvessels or cell membranes and barriers in vivo, which can promote gene penetration. Therefore, the gene transfection device provided in the present application further enables the gene transfection technology of cells to achieve efficient transfection and universality, and has a very broad application prospect.
  • FIG. 1 is a schematic diagram of the overall structure of the gene transfection device provided in the embodiment of the present application.
  • FIG. 2 is an exploded schematic diagram of a partial structure of the gene transfection device provided in the embodiment of the present application.
  • FIG. 3 is a focal spot diagram measured by an ultrasonic sound beam analyzer according to an embodiment of the present application.
  • Figure 4 is a comparison diagram of transfection observed by fluorescence microscope under different experimental conditions.
  • the terms “installed”, “connected” and “connected” should be understood in a broad sense, for example, it may be a fixed connection or a detachable connection Connection, or integral connection; it can be mechanical connection, electrical connection or can communicate with each other; it can be directly connected or indirectly connected through an intermediate medium, it can be the internal communication of two elements or the interaction of two elements relation.
  • installed should be understood in a broad sense, for example, it may be a fixed connection or a detachable connection Connection, or integral connection; it can be mechanical connection, electrical connection or can communicate with each other; it can be directly connected or indirectly connected through an intermediate medium, it can be the internal communication of two elements or the interaction of two elements relation.
  • orientation or positional relationship indicated by the terms “upper”, “lower”, “side”, “inner”, “outer”, “top”, “bottom”, etc. is based on the installation
  • the orientation or positional relationship is only for the convenience of describing the application and simplifying the description, rather than indicating or implying that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation on the application .
  • gene therapy is one of the mainstream research directions in the development of the biotechnology industry.
  • Gene therapy is to introduce foreign genes into target cells in a specific way to correct defective genes or produce corresponding biological effects, so as to achieve the purpose of treating diseases.
  • gene therapy has shown unique advantages in the treatment of major diseases.
  • the three elements of gene therapy are target gene, gene transfection and target cells.
  • gene transfection technology is the key to determine the effect of gene therapy.
  • gene delivery systems that are non-invasive, highly targeted, highly controllable, and can be effectively expressed are particularly important in clinical practice.
  • how to safely and efficiently deliver genes to the target site and stably and persistently express them in tissue cells is a major problem and challenge for gene therapy. Therefore, exploring a new gene transfection method and transfection device has become an urgent task for researchers.
  • viral vectors used for gene transfection can be divided into two categories: viral vectors and non-viral vectors.
  • Common viral vector systems include transcription virus, lentivirus, adenovirus, adeno-associated virus, etc., with high transfection efficiency, but their safety issues, autoimmunity, lack of tumor targeting specificity, and small capacity for carrying the target gene , it is difficult to apply on a large scale, and it is still a bottleneck restricting the further application of viral vectors.
  • Non-viral vectors such as cationic liposomes, provide us with a safer gene delivery method, which has the advantages of low immunogenicity, simple structure, easy chemical modification to change its function, and easy large-scale production and preparation.
  • the gene transfection efficiency using these non-viral vectors is low, and improving the gene transfection efficiency has always been the goal of biomedical researchers.
  • non-viral vectors are increasingly valued by scholars due to their safety advantages.
  • ultrasonic targeted microbubble destruction UTMD
  • the UTMD-mediated gene transfection method provides a new technology for the clinical application of gene therapy, avoiding the defects of previous viral and non-viral vector systems.
  • the principle is to use gas-containing ultrasonic microbubbles to continuously vibrate, expand, shrink, and rupture under the ultrasonic irradiation of a certain sound intensity and mechanical index, resulting in an instantaneous cavitation effect that increases the permeability of the cell membrane and causes it to break down.
  • Temporary and reversible sonopores are generated on the cell membrane, so that the genes bound by microbubbles enter the cells through the sonopores.
  • the cavitation effect and sonoporation effect theory of ultrasound are the main mechanisms by which UTMD promotes and gene transfection. UTMD has been applied to gene transfection studies in various animal experimental disease models, and safe transfection results have been obtained.
  • the existing UTMD ultrasonic transfection apparatus usually uses culture vessels such as culture dishes, culture plates, etc. to carry the complexes of cells, genes and ultrasonic microbubbles, and performs ultrasonic irradiation through a focused ultrasonic probe.
  • Focused ultrasonic probe is a kind of ultrasonic beam that can focus the ultrasonic beam to a small area, and has the characteristics of strong directionality, good penetration, good focusability and good energy precipitation.
  • the sound field distribution of its irradiation is not uniform, the sound field can only cover a part of the culture container, and the cells are usually scattered in various parts of the culture container, that is to say, the ultrasonic irradiation can only cover a part of the cells in the culture container, which leads to Ultrasound probe irradiation energy utilization rate is low, resulting in low gene transfection efficiency.
  • the present application improves the existing ultrasound-mediated gene transfection device, and proposes a gene transfection device that conforms to the ultrasonic sound field, which can realize the conformal coverage of the ultrasonic sound field, thereby realizing the ultrasonic sound field.
  • the maximum utilization of energy thereby improving the efficiency of gene transfection.
  • FIG. 1 is a schematic diagram of the overall structure of the gene transfection device provided in the embodiment of the present application.
  • FIG. 2 is an exploded schematic diagram of a partial structure of the gene transfection device provided in the embodiment of the present application.
  • the gene transfection device provided in the embodiments of the present application can realize conformal coverage of the ultrasonic sound field, and the gene transfection device includes an ultrasonic transducer 1 , an ultrasonic sound field conformer 2 and a focal length adapter 3 .
  • the ultrasonic transducer 1 is used for transmitting ultrasonic waves.
  • the ultrasonic sound field conformer 2 is used for accommodating the complex of genes, cells and ultrasound-responsive particles, and the shape and size of the ultrasonic sound field conformer 2 are determined according to the sound field of the ultrasonic wave.
  • the focal length adapter 3 is used to fix the ultrasonic sound field conformer 2 at a preset position, and the preset position is determined according to the sound field position of the ultrasonic wave.
  • the ultrasonic sound field conformer 2 containing the complexes of genes, cells and ultrasonic responsive particles is fixed at a preset position by the focal length adapter 3, and the ultrasonic transducer 1 can convert electrical signals into ultrasonic signals and convert ultrasonic energy It is transmitted to the ultrasonic sound field conformer 2, and the complex of genes, cells and ultrasonic-responsive particles is subjected to ultrasonic irradiation.
  • the ultrasonic-responsive particles Under the action of the ultrasonic irradiation, the ultrasonic-responsive particles continuously vibrate, expand, contract, and rupture, resulting in an instantaneous cavitation effect that increases the permeability of the cell membrane and produces temporary and reversible sonopores in the cell membrane, so that the gene can Enter the cell through the sonopore to achieve the purpose of gene transfection.
  • the ultrasonic sound field conformer 2 and the ultrasonic transducer 1 are adapted to each other, and the two have a corresponding relationship.
  • the size and shape of the ultrasonic sound field conformer 2 are determined according to the sound field of the ultrasonic waves emitted by the ultrasonic transducer 1, and the ultrasonic sound field conformer 2 is further fixed at a preset position, and the preset position is located in the sound field, And it is determined according to the sound field position of the ultrasonic wave.
  • the ultrasonic sound field conformer 2 and the ultrasonic sound field conform to each other and cover each other, so as to maximize the utilization of ultrasonic energy, thereby improving the gene transfection efficiency.
  • the target transmission characteristics and instantaneous reversible permeability can be improved.
  • Ultrasound irradiation itself has organ targeting, which can improve the local gene transfection efficiency of irradiation. Based on a certain power of ultrasound irradiation, the ultrasound-responsive particles will rupture and release the drugs or genes they carry. Ultrasound responds to particle rupture to produce cavitation effects, including sonoporous effects, microfluidics, etc., which instantly and reversibly increase the permeability of microvessels or cell membranes and barriers in vivo, which can promote gene penetration. Therefore, the gene transfection device provided in the present application further enables the gene transfection technology of cells to achieve efficient transfection and universality, and has a very broad application prospect.
  • the ultrasonic-responsive particles can vibrate, expand, contract, and rupture under the action of ultrasonic waves in response to ultrasonic irradiation, thereby generating an instantaneous cavitation effect.
  • the ultrasonic-responsive particles contained in the ultrasonic sound field conformer 2 may be nano-particles or micro-particles, such as biological nanobubbles.
  • the ultrasound-responsive particles may be ultrasound microbubbles.
  • the ultrasonic microbubbles may be lipid ultrasonic microbubbles, such as phospholipid bubbles, PLGA microbubbles, and the like.
  • the cells accommodated by the ultrasonic sound field conformer 2 may be any one of suspension cells, adherent cells, stem cells or primary cells.
  • the ultrasonic transducer 1 is used to transmit ultrasonic waves.
  • the ultrasonic transducer 1 may be an ultrasonic probe, for example, a focused ultrasonic probe or a planar probe.
  • the ultrasonic frequency range emitted by the ultrasonic transducer 1 can be 0.5-10MHz, for example, 1-3MHZ, 1-5MHZ, 2-3MHZ, 3-7MHZ, 4-8MHZ, 5-10MHZ, etc. 1MHz, 2MHz, 3MHz, 5MHz, 8MHz, 10MHz, etc.
  • the sound intensity range can be 0.1-5W/cm 2 , such as 0.25-3W/cm 2 , 0.5-2W/cm 2 , 3-5W/cm 2 , etc., and can also be 1W/cm 2 , 1.5W /cm 2 , 2.0W/cm 2 , 2.5W/cm 2 , etc.
  • the action time is 0.1 to 10 minutes, such as 0.25 to 3 minutes, 2 to 5 minutes, 3 to 6 minutes, 5 to 8 minutes, etc. In addition, it can also be 1 minute, 1.5 minutes, 4 minutes, 7 minutes , 9 minutes etc.
  • the duty cycle can be 10 ⁇ 50%, for example, 15 ⁇ 45%, 20 ⁇ 40%, 15 ⁇ 30%, 30 ⁇ 50%, etc., and can also be 20%, 25%, 35%, etc. .
  • the transducing material of the ultrasonic transducer 1 includes, but is not limited to, a magnetically compatible material.
  • the ultrasonic transducer 1 can be electrically connected to the ultrasonic signal outputter 9 through a signal transmission line 11 .
  • the ultrasonic signal output device 9 is composed of a power supply, a signal generator and a power amplifier.
  • the electrical signal generated by the signal generator and the power amplifier is transmitted to the ultrasonic transducer 1 through the signal transmission line 11, and the electrical signal is converted into the ultrasonic transducer 1. Ultrasound.
  • the degree of sonoporosity is related to factors such as sound pressure, sound intensity, frequency, working cycle, and irradiation time of ultrasonic irradiation.
  • the working parameters of ultrasonic signal output device 9 can be adjusted according to different experiments or medical needs, so as to achieve energy safety. , and adjustable and controllable.
  • the ultrasonic sound field conformer 2 is used for accommodating the complex of genes, cells and ultrasound-responsive particles, and the shape and size of the ultrasonic sound field conformer 2 are determined according to the sound field of the ultrasonic wave.
  • the shape and size of the ultrasonic sound field conformer 2 may be determined according to the shape, size, energy distribution, etc. of the sound field, which is not limited in this application.
  • the ultrasonic sound field conformer 2 is further fixed at a preset position, and the preset position is determined according to the sound field position of the ultrasonic wave.
  • the preset position may be determined according to the energy distribution of the sound field.
  • the preset position may be located on the central axis of the sound field, which is not limited in this application.
  • FIG. 3 is a focal spot diagram measured by an ultrasonic sound beam analyzer according to an embodiment of the present application.
  • the place where the two bright lines cross each other that is, the area within the black elliptical circle, is the focal spot of the ultrasonic sound field.
  • the focal spot is the area with the highest energy density in the ultrasonic sound field.
  • the focal spot of the sound field can be determined first, and then the ultrasonic sound field conformer 2 is set according to the focal spot. This application does not limit how to determine the focal spot of the sound field.
  • the sound field of the ultrasonic transducer can be simulated by acoustic detection instruments such as hydrophones and sound beam analyzers or based on the physical characteristics of the ultrasonic transducer, the size of the focal spot used can be measured, the volume of the focal spot can be calculated, and the size of the focal spot can be determined. Central location.
  • the shape of the ultrasonic sound field conformer 2 may be determined according to the shape of the focal spot of the sound field.
  • the shape of the ultrasonic sound field conformer 2 may be the same as, or approximately the same as, the shape of the focal spot.
  • the shape of the focal spot generated by the ultrasonic transducer 1 is an ellipsoid, so the shape of the ultrasonic sound field conformer 2 is also set to be an ellipsoid.
  • the shape of the focal spot can be cylindrical, spherical, solid polygon and other shapes.
  • the shape of the ultrasonic sound field conformer 2 can also be set to cylindrical, spherical, solid polygon and other shapes.
  • the shape of the ultrasonic sound field conformer 2 and the shape of the focal spot may also be different.
  • the shape of the focal spot is spherical.
  • the shape of the ultrasonic sound field conformer 2 can be set to an ellipsoid or a three-dimensional polygon, etc. shape, which is not limited in this application.
  • the size of the ultrasonic sound field conformer 2 can be determined according to the size of the focal spot.
  • the size of the ultrasonic sound field conformer 2 may be the same as the size of the focal spot, or similar (approximately the same).
  • the size (or volume) of the ultrasonic sound field conformer 2 may be equal to, slightly larger or slightly smaller than the size (or volume) of the focal spot.
  • the size of the ultrasonic sound field conformer 2 can be proportional to the size of the focal spot, for example, the size of the ultrasonic sound field shaper 2 can be 1.5 times, 2 times, 3 times, 5 times, etc., the size of the focal spot , which is not limited in this application.
  • the focal length adapter 3 is used to fix the ultrasonic sound field conformer 2 at a preset position, and the preset position can be determined according to the position of the focal spot.
  • the preset position may be determined according to the center position of the focal spot.
  • the preset position may be adjacent to the center position of the focal spot, or coincide with the center position of the focal spot.
  • the center of the ultrasonic sound field conformer 2 may be disposed adjacent to the center of the focal spot, or the center of the ultrasonic sound field conformer 2 may coincide with the center of the focal spot.
  • the shape and size of the ultrasonic sound field conformer 2 are the same as the shape and size of the focal spot, and the center position of the ultrasonic sound field conformer 2 and the center position of the focal spot coincide with each other.
  • the ultrasonic sound field conformer 2 is fixed at a preset position through the focal length adapter 3, and the specific form of the focal length adapter 3 is not limited in the present application.
  • the focal length adapter 3 is a collimator.
  • the focal length adapter 3 may also be of other structures, for example, the focal length adapter 3 may be any one of a fixed bracket, an iron wire, a pulling rope, and the like.
  • one end of the focal length adapter 3 is used to connect with the ultrasonic sound field conformer 2, and the other end of the focal length adapter 3 is used to connect with the ultrasonic transducer 1.
  • the ultrasonic sound field conformer 2 is located at the preset position.
  • the focal length adapter 3 provided in the embodiment of the present application can play the role of focusing and positioning.
  • the ultrasonic sound field conformer 2 can be automatically located at the preset position, so there is no need to manually adjust the position of the ultrasonic sound field conformer 2, thereby simplifying the operation steps and ensuring the convenience and efficiency of gene transfection.
  • the ultrasonic sound field conformer 2 is fixedly connected with the focal length adapter 3, and an integrated structure is formed through an integrated molding process.
  • the ultrasonic sound field shaper 2 can be automatically positioned in the preset position. set location. Because there is no need to connect the ultrasonic sound field conformer 2 and the focal length adapter 3, and the two are fixedly connected together without relative displacement, the positioning error caused by the connection of the two is avoided, thereby further simplifying the operation steps , and improve the positioning accuracy.
  • the integral molding process may be injection molding.
  • the ultrasonic sound field conformer 2 and the focal length adapter 3 can also be integrally formed by 3D printing technology.
  • the above two can be integrally formed from photosensitive resin, plastic, rubber, glass, metal and other materials through 3D printing technology.
  • the ultrasonic sound field conformer 2 and the focal length adapter 3 may also be formed of other materials such as glass, metal, plastic, etc., which are not limited in this application.
  • the ultrasonic sound field conformer 2 and the focal length adapter 3 may be detachably connected.
  • the focal length adapter 3 includes a top wall 32 and a peripheral wall 31 .
  • One end of the peripheral wall 31 is connected to the top wall 32 and is arranged around the circumference of the top wall 32 , and the other end of the peripheral wall 31 is provided with The connector 7, the focal length adapter 3 is detachably connected with the ultrasonic transducer 1 through the connector 7.
  • the focal length adapter 3 is detachably connected to the ultrasonic transducer 1 through the connector 7, so that a flexible and simple connection between the two can be realized. At the same time, it is convenient to replace and use different ultrasonic transducers to meet the needs of different experiments or treatments.
  • the connector 7 can be a connection port between the ultrasonic transducer 1 and the focal length adapter 3 , is designed as a chute, is arranged on the inner wall surface of the lower end of the focal length adapter 3 , and can connect with the ultrasonic transducer 1 . Fixed connection at the top.
  • the connector 7 may also be other structures capable of realizing detachable connection between the ultrasonic transducer 1 and the focal length adapter 3 , which is not limited in this application.
  • the ultrasonic sound field conformer 2 is fixedly connected to the inside of the focal length adapter 3 .
  • the ultrasonic sound field conformer 2 is fixedly arranged on the inner wall surface of the top wall 32 , the top wall 32 is provided with a sample loading port 4 , and the sample loading port 4 communicates with the opening of the ultrasonic sound field conformer 2 .
  • the complexes of genes, cells and ultrasound-responsive particles can be put into the ultrasound sound field conformer 2 .
  • the sample loading port 4 can be sealed with a parafilm.
  • the focal length adapter 3 can also be fixedly disposed on the peripheral wall 31 , and the sample loading port 4 is correspondingly opened on the peripheral wall 31 .
  • the sample loading port 4 may not be provided on the focal length adapter 3 , and after the compound is injected into the ultrasonic sound field conformer 2 , the ultrasonic sound field conformer 2 may be fixedly arranged inside the focal length adapter 3 .
  • a concave structure 6 is formed in the middle of the top wall 32 , the height of the inner surface of the concave structure 6 is lower than the height of the inner surface of other parts of the top wall 32 , and the sample loading port 4 is opened in the concave structure 6 .
  • the concave structure 6 By arranging the concave structure 6, on the one hand, it can facilitate feeding and prevent the compound from flowing into the external environment; When the air bubbles exist, the air bubbles will be pushed out to other parts of the top wall 32 , so that the propagation of ultrasonic waves will not be affected by the air bubbles, avoiding the problem of energy attenuation, and thus helping to improve the efficiency of gene transfection. At this time, even if the sample inlet 4 is not provided, the function of removing air bubbles can be achieved.
  • a conductive medium may be filled in the focal length adapter 3, and the conductive medium may be any one of water, coupling fluid or PVA.
  • an exhaust hole 5 may be provided on the top wall 32 , and the air in the focal length adapter 3 may be exhausted through the exhaust hole 5 .
  • the exhaust method is to use a 50ml syringe to inject a medium (most commonly water) until the bubbles disappear before ultrasonic irradiation.
  • the gene transfection device provided in the embodiment of the present application further includes a fixer 8 .
  • the holder 8 is used to fix components such as the ultrasonic transducer 1 , the ultrasonic sound field conformer 2 and the focal length adapter 3 .
  • the fixer 8 may be an iron frame lifting platform, through which the ultrasonic transducer 1 , the ultrasonic sound field conformer 2 and the focal length adapter 3 and other components are fixed above it.
  • a small hole may be opened in the center of the holder 8 to facilitate the passage of the signal transmission line 11 to connect with the ultrasonic transducer 1 .
  • the gene transfection device provided in the embodiment of the present application further includes a medium container 10 , and the medium container 10 contains an ultrasonic wave propagation medium such as water.
  • both the ultrasonic transducer 1 and the ultrasonic sound field conformer 2 include a plurality, and the plurality of ultrasonic transducers 1 and the plurality of ultrasonic sound field conformers 2 are one by one.
  • the sound fields of the ultrasonic waves emitted by each ultrasonic transducer 1 are different from each other.
  • the focal length adapter 3 that matches the ultrasonic transducer 1 can also be further selected for work.
  • the research team of the inventor has used the gene transfection device provided in the examples of the present application for experimental verification, and achieved efficient gene transfection of 293T cells by using the sonoporous effect.
  • the experimental steps are:
  • Figure 4 is a comparison diagram of the transfection observed by fluorescence microscope under different experimental conditions.
  • Figure 4(a) is a blank control group, pure cells, without the complex of phospholipid vesicles and genes, and no cells express mCherry fluorescent protein.
  • Figure 4(b) shows the complex with added phospholipid vesicle + gene, but using the existing gene transfection device for ultrasonic irradiation, only a few cells express fluorescent protein;
  • 4(c) is the addition of phospholipid vesicle +
  • the gene complex was irradiated by ultrasound using the gene transfection device provided in the examples of the present application, and as a result, most of the cells expressed fluorescent proteins.
  • the experimental results prove that the gene transfection device provided in the embodiment of the present application can improve the effect of gene transport and has a higher transfection efficiency.

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Abstract

一种超声声场适形的基因转染装置，包括：超声换能器(1)，用于发射超声波；超声声场适形器(2)，用于容纳基因、细胞和超声响应颗粒的复合物，所述超声声场适形器(2)的形状以及大小根据所述超声波的声场进行确定；焦距适配器(3)，用于将所述超声声场适形器(2)固定于预设位置，所述预设位置根据所述超声波的声场位置进行确定。

Description

一种超声声场适形的基因转染装置
本申请要求于2020年07月27日提交中国专利局、申请号为202010734848.2、申请名称为“一种超声声场适形的基因转染装置”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请涉及生物医学实验中使用的科研装置领域，特别涉及一种超声声场适形的基因转染装置。
 背景技术
基因治疗是通过特定方式将正常基因或有治疗作用的遗传物质导入靶细胞，以纠正基因缺陷或发挥治疗作用，从而达到治疗疾病的目的。基因治疗的三要素是目的基因、基因转染和靶细胞，在靶细胞一定的情况下，基因转染技术是决定基因治疗效果的关键。其中，无创、靶向性强、可控性强，并且能够有效表达的基因传输系统在临床上显得尤为重要。然而，如何将基因安全、高效地输送到靶向部位，且在组织细胞内能够稳定持久表达，是基因治疗面临的重大难题和挑战。
近年来，超声靶向微泡破坏效应(ultrasound targeted microbubble destruction，UTMD)介导的基因转染方法为基因治疗的临床应用提供了一种新的技术。其原理是利用含气的超声微泡，在一定声强和机械指数作用的超声辐照下，不断振动、膨胀、收缩，发生破裂，由此产生瞬间空化效应引起细胞膜通透性增加及在细胞膜上产生暂时的、可逆的声孔，从而与微泡结合的基因能够通过声孔进入细胞内。UTMD作为一种理想的基因转染方法，具有广阔的应用前景。
现有的UTMD仍有其应用缺陷。具体地，现有的UTMD超声转染仪通常使用培养皿、培养板等培养容器来承载细胞、基因和超声微泡的复合物，并且通过聚焦超声探头来进行超声辐照。然而，探头辐照的声场分布不均匀，声场仅能覆盖培养容器的一部分，而细胞通常分散于培养容器的各个部分，由此导致超声探头辐照能量利用率低，进而造成基因转染效率低下。
 技术问题
本申请提供一种超声声场适形的基因转染装置，能够实现对超声声场的适形覆盖，进而实现对超声能量的最大化利用，由此提高了基因转染效率。
 技术解决方案
第一方面，提供了一种超声声场适形的基因转染装置，包括：超声换能器，用于发射超声波；超声声场适形器，用于容纳基因、细胞和超声响应颗粒的复合物，所述超声声场适形器的形状以及大小根据所述超声波的声场进行确定；焦距适配器，用于将所述超声声场适形器固定于预设位置，所述预设位置根据所述超声波的声场位置进行确定。
根据本申请实施例提供的基因转染装置，超声声场适形器与超声换能器是相互适配使用的，二者具有对应关系。超声声场适形器的大小和形状是根据超声换能器所发射的超声波的声场进行确定的，超声声场适形器进一步被固定于预设位置，该预设位置位于该声场内，并且是根据超声波的声场位置进行确定的，超声声场适形器与超声声场相互适形覆盖，进而能够实现对超声能量的最大化利用，由此提高了基因转染效率。
此外，根据本申请实施例提供的基因转染装置，能够提高靶向传输特性和瞬间可逆渗透性。在超声辐照本身具有器官靶向性，可使辐照局部基因转染效率提高靶向部位基于一定功率的超声辐照，超声响应颗粒发生破裂，释放所携带的药物或基因。超声响应颗粒破裂产生空化效应，包括声孔效应、微射流等使微血管或细胞膜及体内屏障产生瞬间可逆的通透性升高，可以促进基因的渗透。因此，本申请提供的基因转染装置进一步使细胞的基因转染技术实现高效转染以及普遍性，具有非常广阔的应用前景。
可选地，超声声场适形器所容纳的超声响应颗粒可以为纳米颗粒或者微米颗粒，例如生物纳米泡等。
可选地，该超声响应颗粒可以为超声微泡。
例如，该超声微泡可以为脂质超声微泡，例如磷脂泡、PLGA微泡等。
可选地，超声声场适形器所容纳的细胞可以为悬浮细胞、贴壁细胞、干细胞或者原代细胞等中的任意一种。
在一种可能的设计中，所述超声声场适形器的形状根据所述声场的焦斑的形状进行确定，所述超声声场适形器的大小根据所述焦斑的大小进行确定，所述预设位置根据所述焦斑的位置进行确定。
在一种可能的设计中，所述超声声场适形器的形状与所述焦斑的形状相同。
在一种可能的设计中，所述超声声场适形器的大小与所述焦斑的大小相同。
在一种可能的设计中，所述预设位置为所述焦斑的中心位置。
在一种可能的设计中，所述焦距适配器的一端用于与所述超声声场适形器相连接，所述焦距适配器的另一端用于与所述超声换能器相连接，当所述超声声场适形器、所述焦距适配器与所述超声换能器连接到一起时，所述超声声场适形器位于所述预设位置。
也就是说，本申请实施例提供的焦距适配器能够起到聚焦定位的作用，当超声声场适形器、焦距适配器与超声换能器三者连接到一起时，超声声场适形器能够自动位于该预设位置，因此不需要手动调整超声声场适形器的位置，由此简化了操作步骤，保证了基因转染的便捷和高效性。
在一种可能的设计中，所述超声声场适形器与所述焦距适配器通过一体成型工艺形成一体结构。通过以上设置，超声声场适形器与焦距适配器二者之间不会发生相对位移，当焦距适配器被连接于超声换能器之上时，超声声场适形器能够自动位于预设位置。由于不需要对超声声场适形器与焦距适配器进行连接操作，并且二者固定连接在一起不会发生相对位移，避免了上述二者进行连接造成的定位误差，由此进一步简化了操作步骤，并且提高了定位的准确性。
在一种可能的设计中，所述超声声场适形器与所述焦距适配器通过3D打印技术一体成型制成。
例如，可以由光敏树脂、塑料、橡胶、玻璃、金属等通过3D打印技术一体成型制成。
在一种可能的设计中，所述超声声场适形器固定连接于所述焦距适配器的内部。
在一种可能的设计中，所述焦距适配器包括顶壁，所述超声声场适形器固定设置于所述顶壁的内壁面上，所述顶壁上开设有上样口，所述上样口与所述超声声场适形器的开口相连通。
在一种可能的设计中，所述顶壁上开设有排气孔。
在一种可能的设计中，所述顶壁的中部形成内凹结构，所述内凹结构的内表面的高度低于顶壁其他部分内表面的高度，所述上样口开设于所述内凹结构内。
通过设置内凹结构，一方面能够方便投料，防止复合物被流入外部环境中；另一方面，内凹结构的内表面的高度低于顶壁其他部分内表面的高度，这样当有气泡存在时，气泡将被排挤到顶壁的其他部分，由此使得超声波的传播不会受气泡影响，避免能量衰减这一问题，进而有利于提高基因转染的效率。此时，即使不设置上样口同样也能起到排除气泡的作用。
在一种可能的设计中，所述焦距适配器还包括周壁，所述周壁的一端连接于所述顶壁上并且环绕所述顶壁的周向设置，所述周壁的另一端设置有连接器，所述焦距适配器通过所述连接器与所述超声换能器可拆卸连接。焦距适配器通过所述连接器与超声换能器可拆卸连接，可以实现二者灵活简便的连接。同时也方便更换使用不同的超声换能器，以满足不同的实验或者治疗的需求。
在一种可能的设计中，所述超声换能器为聚焦式超声探头。
在一种可能的设计中，所述超声响应颗粒为超声微泡。
在一种可能的设计中，所述超声换能器和超声声场适形器均包括多个，多个所述超声换能器与多个所述超声声场适形器一一对应，各个超声换能器所发射的超声波的声场各不相同。
这样，使用时，可以首先根据实验和医疗需求确定选择哪一个超声换能器来进行工作，在确定了超声换能器之后，可以进一步选择与之匹配的超声声场适形器来进行工作。
 有益效果
根据本申请实施例提供的基因转染装置，超声声场适形器与超声换能器是相互适配使用的，二者具有对应关系。超声声场适形器的大小和形状是根据超声换能器所发射的超声波的声场进行确定的，超声声场适形器进一步被固定于预设位置，该预设位置位于该声场内，并且是根据超声波的声场位置进行确定的，超声声场适形器与超声声场相互适形覆盖，进而能够实现对超声能量的最大化利用，由此提高了基因转染效率。
此外，根据本申请实施例提供的基因转染装置，能够提高靶向传输特性和瞬间可逆渗透性。在超声辐照本身具有器官靶向性，可使辐照局部基因转染效率提高靶向部位基于一定功率的超声辐照，超声响应颗粒发生破裂，释放所携带的药物或基因。超声响应颗粒破裂产生空化效应，包括声孔效应、微射流等使微血管或细胞膜及体内屏障产生瞬间可逆的通透性升高，可以促进基因的渗透。因此，本申请提供的基因转染装置进一步使细胞的基因转染技术实现高效转染以及普遍性，具有非常广阔的应用前景。
 附图说明
图1是本申请实施例提供的基因转染装置的整体结构示意图。
图2是本申请实施例提供的基因转染装置的部分结构的分解示意图。
图3是本申请实施例提供的使用超声声束分析仪测量的焦斑图。
图4是不同实验条件下荧光显微镜观察转染情况的对比图。
 本发明的实施方式
下面将结合附图，对本申请中的技术方案进行描述。显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。
在本申请的描述中，需要说明的是，除非另有明确的规定和限定，术语“安装”、“相连”、“连接”应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或一体地连接；可以是机械连接，也可以是电连接或可以相互通讯；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通或两个元件的相互作用关系。对于本领域的普通技术人员而言，可以根据具体情况理解上述术语在本申请中的具体含义。
在本申请的描述中，需要理解的是，术语“上”、“下”、 “侧”、“内”、“外”、“顶”、“底”等指示的方位或位置关系为基于安装的方位或位置关系，仅是为了便于描述本申请和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本申请的限制。
还需说明的是，本申请实施例中以同一附图标记表示同一组成部分或同一零部件，对于本申请实施例中相同的零部件，图中可能仅以其中一个零件或部件为例标注了附图标记，应理解的是，对于其他相同的零件或部件，附图标记同样适用。
基因治疗作为生物技术新的里程碑，是生物技术产业发展的主流研究方向之一。基因治疗是通过特定的方式将外源基因导入靶细胞，以纠正缺陷的基因或产生相应的生物学效应，从而达到治疗疾病目的。近年来，随着分子生物学的发展，基因治疗在重大疾病治疗中显示了独特的优势。研究表明，基因治疗的三要素是目的基因、基因转染和靶细胞，在靶细胞一定的情况下，基因转染技术是决定基因治疗效果的关键。其中，无创、靶向性强、可控性强，并且能够有效表达的基因传输系统在临床上显得尤为重要。然而，如何将基因安全、高效地输送到靶向部位，且在组织细胞内能够稳定持久表达，是基因治疗面临的重大难题和挑战。因此，探索一种新的基因转染方法和转染装置已成为研究者们紧迫的任务。
目前，用于基因转染的载体可分为两大类：病毒载体和非病毒载体。常见的病毒载体系统有转录病毒、慢病毒、腺病毒、腺相关病毒等，转染效率较高，但其安全性问题、自身免疫原性、缺乏肿瘤靶向特异性，携载目的基因容量小，难于大规模应用，仍是制约病毒载体进一步应用的瓶颈。非病毒载体，如阳离子脂质体等为我们提供了更为安全的基因运输方法，具有免疫原性低、结构简单，易于化学修饰来改变其功能，易于规模化生产和制备等优势。但是采用这些非病毒载体的基因转染效率较低，提高基因转染效率一直是生物医学研究者探索的目标。相对于大多数的病毒载体来说，非病毒载体以安全性的优势日益受到学者们的重视。
近年来研究显示，利用超声微泡作为载体，同时进行超声照射可靶向转染目的基因，产生一定的生物学效应，称之为超声靶向微泡破坏效应(ultrasound targeted microbubble destruction，UTMD)介导的基因转染法。UTMD介导的基因转染方法为基因治疗的临床应用提供了一种新的技术，避免了以往病毒及非病毒载体系统的缺陷。其原理是利用含气的超声微泡，在一定声强和机械指数作用的超声辐照下，不断振动、膨胀、收缩，发生破裂，由此产生瞬间空化效应引起细胞膜通透性增加及在细胞膜上产生暂时的、可逆的声孔，从而微泡结合的基因通过声孔进入细胞内，这种超声的空化效应和声孔效应理论是UTMD促进与基因转染的主要机制。现已有将UTMD应用于多种动物实验疾病模型的基因转染研究，取得了安全的转染结果。
然而，现有的UTMD仍有其应用缺陷。具体地，现有的UTMD超声转染仪通常使用培养皿、培养板等培养容器来承载细胞、基因和超声微泡的复合物，并且通过聚焦超声探头来进行超声辐照。聚焦超声探头是一种能够将超声波束聚焦到较小的区域，具有方向性强、穿透性好、可聚性好和良好的能量沉淀性等特点。但是其辐照的声场分布不均匀，声场仅能覆盖培养容器的一部分，而细胞通常分散于培养容器的各个部分，也就是说，超声辐照仅能覆盖培养容器中的一部分细胞，由此导致超声探头辐照能量利用率低，进而造成基因转染效率低下。
因此，针对上述缺陷，本申请对现有的超声介导的基因转染装置进行改进，提出一种超声声场适形的基因转染装置，能够实现对超声声场的适形覆盖，进而实现对超声能量的最大化利用，由此提高了基因转染效率。
图1是本申请实施例提供的基因转染装置的整体结构示意图。图2是本申请实施例提供的基因转染装置的部分结构的分解示意图。
如图1、2所示，本申请实施例提供的基因转染装置能够实现对超声声场的适形覆盖，该基因转染装置包括超声换能器1、超声声场适形器2和焦距适配器3。
其中，超声换能器1用于发射超声波。超声声场适形器2用于容纳基因、细胞和超声响应颗粒的复合物，超声声场适形器2的形状以及大小根据超声波的声场进行确定。焦距适配器3用于将超声声场适形器2固定于预设位置，该预设位置根据超声波的声场位置进行确定。
具体地，容纳基因、细胞和超声响应颗粒的复合物的超声声场适形器2通过焦距适配器3被固定于预设位置，超声换能器1可以将电信号转化为超声信号，并将超声能量传送到超声声场适形器2内，对基因、细胞和超声响应颗粒的复合物进行超声辐照作用。在该超声辐照作用下，超声响应颗粒不断振动、膨胀、收缩，发生破裂，由此产生瞬间空化效应引起细胞膜通透性增加及在细胞膜上产生暂时的、可逆的声孔，从而基因可以通过声孔进入细胞内，实现基因转染的目的。
根据本申请实施例提供的基因转染装置，超声声场适形器2与超声换能器1是相互适配使用的，二者具有对应关系。超声声场适形器2的大小和形状是根据超声换能器1所发射的超声波的声场进行确定的，超声声场适形器2进一步被固定于预设位置，该预设位置位于该声场内，并且是根据超声波的声场位置进行确定的，超声声场适形器2与超声声场相互适形覆盖，进而能够实现对超声能量的最大化利用，由此提高了基因转染效率。
此外，根据本申请实施例提供的基因转染装置，能够提高靶向传输特性和瞬间可逆渗透性。在超声辐照本身具有器官靶向性，可使辐照局部基因转染效率提高靶向部位基于一定功率的超声辐照，超声响应颗粒发生破裂，释放所携带的药物或基因。超声响应颗粒破裂产生空化效应，包括声孔效应、微射流等使微血管或细胞膜及体内屏障产生瞬间可逆的通透性升高，可以促进基因的渗透。因此，本申请提供的基因转染装置进一步使细胞的基因转染技术实现高效转染以及普遍性，具有非常广阔的应用前景。
在本申请实施例中，超声响应颗粒可以响应于超声辐照，在超声波的作用下能够发生振动、膨胀、收缩，发生破裂，由此产生瞬间空化效应。
可选地，超声声场适形器2所容纳的超声响应颗粒可以为纳米颗粒或者微米颗粒，例如生物纳米泡等。
可选地，该超声响应颗粒可以为超声微泡。
例如，该超声微泡可以为脂质超声微泡，例如磷脂泡、PLGA微泡等。
可选地，超声声场适形器2所容纳的细胞可以为悬浮细胞、贴壁细胞、干细胞或者原代细胞等中的任意一种。
超声换能器1用于发射超声波。可选地，超声换能器1可以为超声探头，例如可以为聚焦式超声探头或平面探头等。
可选地，超声换能器1发射的超声波频率范围可以为0.5～10MHz，例如1~3MHZ，1~5MHZ，2~3MHZ，3~7MHZ，4~8MHZ，5~10MHZ等，此外，还可以为1MHz，2MHz，3MHz，5MHz，8MHz，10MHz等。
可选地，声强范围可以为0.1～5W/cm 2，例如0.25～3W/cm 2，0.5～2W/cm 2，3～5W/cm 2等，此外还可以为1W/cm 2，1.5W/cm 2，2.0W/cm 2，2.5W/cm 2等。
可选地，作用时间为0.1～10分钟，例如0.25～3分钟，2~5分钟，3~6分钟，5~8分钟等，此外，还可以为1分钟，1.5分钟，4分钟，7分钟，9分钟等。
可选地，占空比可以为10~50%，例如，15~45%，20~40%，15~30%，30~50%等，此外还可以为20%，25%，35%等。
应理解，上述参数仅作为示例，本申请所提供的超声换能器1不限于其他参数。
再例如，超声换能器1的换能材料包括但不限于磁兼容材料。
超声换能器1可以通过信号传输线11与超声信号输出器9电连接。超声信号输出器9由电源、信号发生器及功率放大器组成，信号发生器及功率放大器产生的电信号通过信号传输线11输送到超声换能器1上，电信号在超声换能器1上转化为超声波。
声孔效应的程度与超声辐照的声压、声强、频率、工作周期、辐照时间等因素有关，根据不同实验或者医疗需求可以调节超声信号输出器9的工作参数，进而能够实现能量安全、且可调可控。
超声声场适形器2用于容纳基因、细胞和超声响应颗粒的复合物，超声声场适形器2的形状以及大小根据超声波的声场进行确定。可选地，可以根据声场的形状、大小、能量分布等确定超声声场适形器2的形状以及大小，本申请对此不做限定。
超声声场适形器2进一步被固定于预设位置，该预设位置是根据超声波的声场位置进行确定的。可选地，该预设位置可以是根据声场的能量分布情况确定的。例如，该预设位置可以位于声场的中轴线上，本申请对此不做限定。
图3是本申请实施例提供的使用超声声束分析仪测量的焦斑图。在图3中，两条亮线相互交叉的地方，也即黑色椭圆圈内的区域，即为超声波声场的焦斑。焦斑是超声波声场中能量密度最大的区域，在本申请实施例中，可以首先确定声场的焦斑，之后根据焦斑来设置超声声场适形器2。本申请对如何确定声场的焦斑不做限定。
例如，可以通过水听器、声束分析仪等声学检测仪器或根据超声换能器的物理特征对其声场进行计算机仿真，测量所用焦斑的大小，并计算焦斑的体积、确定焦斑的中心位置。
可选地，超声声场适形器2的形状可以根据声场的焦斑的形状进行确定。例如，超声声场适形器2的形状可以和焦斑的形状相同，或者近似相同。
如图2所示，在本申请实施例中，超声换能器1产生的焦斑的形状为椭球形，因此超声声场适形器2的形状也被设置成椭球形。
在其他实施方式中，焦斑的形状可以为圆柱形、球形、立体多边形等其他形状，相对应的，也可以将超声声场适形器2的形状设置为圆柱形、球形、立体多边形等形状。
应理解，超声声场适形器2的形状和焦斑的形状也可以不相同，例如，焦斑的形状为球形，此时可以将超声声场适形器2的形状设置为椭球形或者立体多边形等形状，本申请对此不做限定。
超声声场适形器2的大小可以根据焦斑的大小进行确定。例如，超声声场适形器2的大小可以和焦斑的大小相同，或者相近（近似相同）。
可选地，超声声场适形器2的尺寸（或者体积）可以等于、略大于或者略小于焦斑的尺寸（或者体积）。
可选地，超声声场适形器2的大小和焦斑的大小可以成比例，例如，超声声场适形器2的大小可以为焦斑的大小的1.5倍、2倍、3倍、5倍等，本申请对此不作限定。
焦距适配器3用于将超声声场适形器2固定于预设位置，该预设位置可以根据焦斑的位置进行确定。
可选地，该预设位置可以根据焦斑的中心位置进行确定。例如，该预设位置可以邻近焦斑的中心位置，或者，和焦斑的中心位置重合。
也就是说，超声声场适形器2的中心可以临近焦斑的中心位置进行设置，或者，超声声场适形器2的中心可以和焦斑的中心相互重合。
在本申请实施例中，超声声场适形器2的形状和焦斑的形状相同，大小相同，并且超声声场适形器2的中心位置和焦斑的中心位置相互重合。通过以上设置，能够最大化利用超声能量，进而能够提高转染效率。
在本申请实施例中，通过焦距适配器3将超声声场适形器2固定于预设位置，本申请对焦距适配器3的具体形式不作限定。在本申请实施例中，焦距适配器3为准直器。
可选地，在其他实施方式中，焦距适配器3也可以为其他结构，例如，该焦距适配器3可以为固定支架、铁丝、拉绳等中的任意一种。
如图1、2所示，在本申请实施例中，焦距适配器3的一端用于与超声声场适形器2相连接，焦距适配器3的另一端用于与超声换能器1相连接，当超声声场适形器2、焦距适配器3与超声换能器1连接到一起时，超声声场适形器2位于该预设位置。
也就是说，本申请实施例提供的焦距适配器3能够起到聚焦定位的作用，当超声声场适形器2、焦距适配器3与超声换能器1三者连接到一起时，超声声场适形器2能够自动位于该预设位置，因此不需要手动调整超声声场适形器2的位置，由此简化了操作步骤，保证了基因转染的便捷和高效性。
在本申请实施例中，超声声场适形器2与焦距适配器3固定连接，并且通过一体成型工艺形成一体结构。
通过以上设置，超声声场适形器2与焦距适配器3二者之间不会发生相对位移，当焦距适配器3被连接于超声换能器1之上时，超声声场适形器2能够自动位于预设位置。由于不需要对超声声场适形器2与焦距适配器3进行连接操作，并且二者固定连接在一起不会发生相对位移，避免了上述二者进行连接造成的定位误差，由此进一步简化了操作步骤，并且提高了定位的准确性。
例如，该一体成型工艺可以为注塑成型。
再例如，超声声场适形器2与所述焦距适配器3也可以通过3D打印技术一体成型制成。此时，可以上述二者可以由光敏树脂、塑料、橡胶、玻璃、金属等材料通过3D打印技术一体成型。
可选地，在其他实施方式中，超声声场适形器2与所述焦距适配器3也可以由玻璃、金属、塑胶等其他材料构成，本申请对此不做限定。
可选地，在其他实施方式中，超声声场适形器2和焦距适配器3可以为可拆卸连接。
如图2所示，在本申请实施例中，焦距适配器3包括顶壁32周壁31，周壁31的一端连接于顶壁32上并且环绕顶壁32的周向设置，周壁31的另一端设置有连接器7，焦距适配器3通过所述连接器7与超声换能器1可拆卸连接。
焦距适配器3通过所述连接器7与超声换能器1可拆卸连接，可以实现二者灵活简便的连接。同时也方便更换使用不同的超声换能器，以满足不同的实验或者治疗的需求。
在本申请实施例中，连接器7可以为超声换能器1与焦距适配器3的连接端口，为滑槽设计，设置于焦距适配器3的下端内壁面上，并且能够和超声换能器1的上端固定连接。在其他实施方式中，连接器7也可以为能够实现超声换能器1和焦距适配器3的可拆卸连接的其他结构，本申请对此不作限定。
如图2所示，超声声场适形器2固定连接于焦距适配器3的内部。具体地，超声声场适形器2被固定设置于顶壁32的内壁面上，顶壁32上开设有上样口4，上样口4与超声声场适形器2的开口相连通。从而能够将基因、细胞和超声响应颗粒的复合物投放入超声声场适形器2内。
此时，由于超声声场适形器2不是密封的，在使用期间，为了避免细胞污染，可以用封口膜密封上样口4。
在其他实施方式中，焦距适配器3也可以被固定设置于周壁31上，并且相应的将上样口4开设于周壁31上。
在其他实施方式中，也可以不在焦距适配器3上开设上样口4，可以将复合物注入超声声场适形器2内部之后，在将超声声场适形器2固定设置于焦距适配器3的内部。
进一步地，如图1、2所示，
顶壁32的中部形成内凹结构6，内凹结构6的内表面的高度低于顶壁32其他部分内表面的高度，上样口4开设于内凹结构6内。
通过设置内凹结构6，一方面能够方便投料，防止复合物被流入外部环境中；另一方面，内凹结构6的内表面的高度低于顶壁32其他部分内表面的高度，这样当有气泡存在时，气泡将被排挤到顶壁32的其他部分，由此使得超声波的传播不会受气泡影响，避免能量衰减这一问题，进而有利于提高基因转染的效率。此时，即使不设置上样口4同样也能起到排除气泡的作用。
进一步地，为了传导超声波，可以在焦距适配器3内填充传导介质，该传导介质可以为水、耦合液或PVA等中的任意一种。
进一步地，为了使超声波传播不受气泡影响，避免能量衰减，可以在顶壁32上开设排气孔5，通过排气孔5来将焦距适配器3内的空气排出。
例如，在使用前，应该检查焦距适配器3内部是否存在气泡，如果有气泡，则应通过排气孔5进行排气。排气方法为使用50ml注射器注入介质（最常用为水），直到气泡消失，方可进行超声辐照。
如图1所示，本申请实施例提供的基因转染装置还包括固定器8。固定器8用于固定超声换能器1、超声声场适形器2以及焦距适配器3等部件。
可选地，固定器8可以为铁架升降台，通过铁架升降台使超声换能器1、超声声场适形器2以及焦距适配器3等部件固定于其上方。
可选地，固定器8的中心可以开设小孔，方便信号传输线11穿过与超声换能器1相连接。
如图1所示，本申请实施例提供的基因转染装置还包括介质容器10，介质容器10盛有水等超声波传播介质。
为了满足不同实验和医疗需求，在本申请实施例中，超声换能器1和超声声场适形器2均包括多个，多个超声换能器1与多个超声声场适形器2一一对应，各个超声换能器1所发射的超声波的声场各不相同。
这样，使用时，可以首先根据实验和医疗需求确定选择哪一个超声换能器1来进行工作，在确定了超声换能器1之后，可以进一步选择与之匹配的超声声场适形器2来进行工作。
可选地，当超声声场适形器2和焦距适配器3为一体结构时，在确定了超声换能器1之后，也可以进一步选择与之匹配的焦距适配器3来进行工作。
发明人所在研究团队已经利用本申请实施例供的基因转染装置进行实验验证，利用声孔效应实现293T细胞高效基因转染。实验步骤为：
1、提前准备基因转染装置，将固定器8置于介质容器10内的水中，把超声换能器1置于其上，并嵌合相应的焦距适配器3，通过排气孔5排除气泡。启动超声信号输出器9，将其转染参数设置为1~3Mhz频率，10-50%占空比，0.1-10分钟辐照时间。
2、使用颗粒计数分析仪计算制备好的磷脂泡数量；
3、每孔取5*10 5-5*10 8磷脂泡+0.5-5ug质粒混匀制作成转染的复合物，室温静置10-30分钟；
4、将细胞消化，计数每孔10-20*10 4细胞，加入制作好的磷脂泡和质粒的复合物，通过焦距适配器3上开设上样口4将复合物放入超声声场适形器2内，通过封口膜密封上样口4，之后按照实验设计的超声参数进行辐照，注意无菌操作。
5、操作完毕后，把细胞从超声声场适形器2中回收，尽快加入DMEM培养基，铺在24孔板，孵育24h，24h后用荧光显微镜观察转染情况。最终本次实验的结果如图4（c）所示。
为了对本申请实施例提供的基因转染装置的性能有个更加清楚的了解，发明人所在研究团队还做了对比实验，图4是不同实验条件下荧光显微镜观察转染情况的对比图。
其中，图4（a）为空白对照组，单纯细胞，不加磷脂泡和基因的复合物，结果没有细胞表达mCherry荧光蛋白。图4（b）为加了磷脂泡+基因的复合物，但是使用现有的基因转染装置进行超声辐照，结果仅有极少数细胞表达荧光蛋白；4（c）为加了磷脂泡+基因的复合物，且使用本申请实施例提供的基因转染装置进行超声辐照，结果大部分细胞表达荧光蛋白。实验结果证明，本申请实施例提供的基因转染装置可以提高基因运输效果，具有较高的转染效率。
以上所述，仅为本申请的具体实施方式，但本申请的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本申请揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本申请的保护范围之内。因此，本申请的保护范围应以所述权利要求的保护范围为准。

Claims (16)

  1. 一种超声声场适形的基因转染装置，其特征在于，包括：
    超声换能器（1），用于发射超声波；
    超声声场适形器（2），用于容纳基因、细胞和超声响应颗粒的复合物，所述超声声场适形器（2）的形状以及大小根据所述超声波的声场进行确定；
    焦距适配器（3），用于将所述超声声场适形器（2）固定于预设位置，所述预设位置根据所述超声波的声场位置进行确定。
  2. 根据权利要求1所述的基因转染装置，其特征在于，所述超声声场适形器（2）的形状根据所述声场的焦斑的形状进行确定，所述超声声场适形器（2）的大小根据所述焦斑的大小进行确定，所述预设位置根据所述焦斑的位置进行确定。
  3. 根据权利要求2所述的基因转染装置，其特征在于，所述超声声场适形器（2）的形状与所述焦斑的形状相同。
  4. 根据权利要求2或3所述的基因转染装置，其特征在于，所述超声声场适形器（2）的大小与所述焦斑的大小相同。
  5. 根据权利要求2-4中任一项所述的基因转染装置，其特征在于，所述预设位置为所述焦斑的中心位置。
  6. 根据权利要求1-5中任一项所述的基因转染装置，其特征在于，所述焦距适配器（3）的一端用于与所述超声声场适形器（2）相连接，所述焦距适配器（3）的另一端用于与所述超声换能器（1）相连接，当所述超声声场适形器（2）、所述焦距适配器（3）与所述超声换能器（1）连接到一起时，所述超声声场适形器（2）位于所述预设位置。
  7. 根据权利要求6所述的基因转染装置，其特征在于，所述超声声场适形器（2）与所述焦距适配器（3）通过一体成型工艺形成一体结构。
  8. 根据权利要求7所述的基因转染装置，其特征在于，所述超声声场适形器（2）与所述焦距适配器（3）通过3D打印技术一体成型制成。
  9. 根据权利要求6-8中任一项所述的基因转染装置，其特征在于，所述超声声场适形器（2）固定连接于所述焦距适配器（3）的内部。
  10. 根据权利要求9所述的基因转染装置，其特征在于，所述焦距适配器（3）包括顶壁（32），所述超声声场适形器（2）固定设置于所述顶壁（32）的内壁面上，所述顶壁（32）上开设有上样口（4），所述上样口（4）与所述超声声场适形器（2）的开口相连通。
  11. 根据权利要求10所述的基因转染装置，其特征在于，所述顶壁（32）上开设有排气孔（5）。
  12. 根据权利要求10或11所述的基因转染装置，其特征在于，所述顶壁（32）的中部形成内凹结构（6），所述内凹结构（6）的内表面的高度低于所述顶壁（32）其他部分内表面的高度，所述上样口（4）开设于所述内凹结构（6）上。
  13. 根据权利要求10-12中任一项所述的基因转染装置，其特征在于，所述焦距适配器（3）还包括周壁（31），所述周壁（31）的一端连接于所述顶壁（32）上并且环绕所述顶壁（32）的周向设置，所述周壁（31）的另一端设置有连接器（7），所述焦距适配器（3）通过所述连接器（7）与所述超声换能器（1）可拆卸连接。
  14. 根据权利要求1-13中任一项所述的基因转染装置，其特征在于，所述超声换能器（1）为聚焦式超声探头。
  15. 根据权利要求1-14中任一项所述的基因转染装置，其特征在于，所述超声响应颗粒为超声微泡。
  16. 根据权利要求1-15中任一项所述的基因转染装置，其特征在于，所述超声换能器（1）和超声声场适形器（2）均包括多个，多个所述超声换能器（1）与多个所述超声声场适形器（2）一一对应，各个超声换能器（1）所发射的超声波的声场各不相同。
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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012030675A1 (en) * 2010-08-28 2012-03-08 The Trustees Of Columbia University In The City Of New York Systems, methods, and devices for plasmid gene transfection using polymer-modified microbubbles
CN202610237U (zh) * 2012-06-18 2012-12-19 陈智毅 有利于超声介导基因转染的操作平台
CN202643683U (zh) * 2012-07-12 2013-01-02 陈智毅 一种用于utmd介导基因转导的装置
CN106031795A (zh) * 2016-07-22 2016-10-19 浙江大学 一种超声微泡转载装置及其方法
CN106868049A (zh) * 2016-12-29 2017-06-20 天津大学 一种导入装置及导入方法
CN208218882U (zh) * 2018-04-10 2018-12-11 华中科技大学同济医学院附属协和医院 一种联合超声靶向微泡破坏技术介导基因转染的装置
CN208485894U (zh) * 2018-06-27 2019-02-12 北京大学深圳医院 超声微泡介导转染用装置
CN111808746A (zh) * 2020-07-27 2020-10-23 中国科学院深圳先进技术研究院 一种超声声场适形的基因转染装置

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012030675A1 (en) * 2010-08-28 2012-03-08 The Trustees Of Columbia University In The City Of New York Systems, methods, and devices for plasmid gene transfection using polymer-modified microbubbles
CN202610237U (zh) * 2012-06-18 2012-12-19 陈智毅 有利于超声介导基因转染的操作平台
CN202643683U (zh) * 2012-07-12 2013-01-02 陈智毅 一种用于utmd介导基因转导的装置
CN106031795A (zh) * 2016-07-22 2016-10-19 浙江大学 一种超声微泡转载装置及其方法
CN106868049A (zh) * 2016-12-29 2017-06-20 天津大学 一种导入装置及导入方法
CN208218882U (zh) * 2018-04-10 2018-12-11 华中科技大学同济医学院附属协和医院 一种联合超声靶向微泡破坏技术介导基因转染的装置
CN208485894U (zh) * 2018-06-27 2019-02-12 北京大学深圳医院 超声微泡介导转染用装置
CN111808746A (zh) * 2020-07-27 2020-10-23 中国科学院深圳先进技术研究院 一种超声声场适形的基因转染装置

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DONG GUIFANG, RAN HAI-TAO, WANG ZHI-GANG, GONG YU-PING, WANG XI: "Targeted Microbubbles Destruction With Low Intensity Focused Ultrasound", ZHONGGUO JIERU YINGXIANG YU ZHILIAOXUE = CHINESE JOURNAL OF INTERVENTIONAL IMAGING AND THERAPY, CN, vol. 10, no. 3, 31 December 2013 (2013-12-31), CN , pages 171 - 174, XP055892020, ISSN: 1672-8475, DOI: 10.13929/j.1672-8475.2013.03.005 *

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