CN212404115U

CN212404115U - Gene transfection device with shape-adapted ultrasonic sound field

Info

Publication number: CN212404115U
Application number: CN202021510276.1U
Authority: CN
Inventors: 严飞; 蒋天安; 谢丽婷; 邹俊杰
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2020-07-27
Filing date: 2020-07-27
Publication date: 2021-01-26
Anticipated expiration: 2030-07-27

Abstract

The application provides an ultrasonic acoustic field conformal gene transfection device, which comprises: an ultrasonic transducer (1) for emitting ultrasonic waves; an ultrasonic field shaper (2) for containing a complex of genes, cells and ultrasound responsive particles, the shape and size of the ultrasonic field shaper (2) being determined from the sound field of the ultrasonic waves; the focal length adapter (3) is used for fixing the ultrasonic sound field adapter (2) at a preset position, and the preset position is determined according to the sound field position of the ultrasonic wave. The gene transfection device provided by the application can realize conformal coverage on an ultrasonic sound field, further realize maximum utilization of ultrasonic energy, and further improve gene transfection efficiency.

Description

Gene transfection device with shape-adapted ultrasonic sound field

Technical Field

The application relates to the field of scientific research devices used in biomedical experiments, in particular to an ultrasonic sound field conformal gene transfection device.

Background

Gene therapy is the treatment of disease by introducing normal genes or therapeutically useful genetic material into target cells in a specific manner to correct gene defects or to exert therapeutic effects. The three elements of gene therapy are target gene, gene transfection and target cell, and under certain conditions of the target cell, the gene transfection technology is the key for determining the gene therapy effect. Among them, the gene delivery system which is noninvasive, highly targeted, highly controllable, and capable of effectively expressing is of great importance in clinical practice. However, how to deliver genes to a target site safely and efficiently and to enable stable and persistent expression in tissue cells is a significant challenge and challenge for gene therapy.

In recent years, an ultrasonic targeted micro bubble destruction (UTMD) mediated gene transfection method provides a new technology for clinical application of gene therapy. The principle is that the gas-containing ultrasonic microvesicles are continuously vibrated, expanded and contracted to be broken under the ultrasonic irradiation with certain sound intensity and mechanical index action, so that instant cavitation effect is generated to increase the permeability of cell membranes and generate temporary and reversible sound holes on the cell membranes, and genes combined with the microvesicles can enter cells through the sound holes. UTMD has wide application prospect as an ideal gene transfection method.

Existing UTMDs still have their drawbacks in use. Specifically, existing UTMD ultrasonic transfectants generally use culture vessels such as petri dishes and plates to carry a complex of cells, genes, and ultrasonic microbubbles, and perform ultrasonic irradiation by a focused ultrasonic probe. However, the sound field radiated by the probe is not uniformly distributed, the sound field can only cover one part of the culture container, and the cells are usually scattered in each part of the culture container, thereby causing low energy utilization rate of the ultrasonic probe radiation, and further causing low gene transfection efficiency.

SUMMERY OF THE UTILITY MODEL

The application provides a gene transfection device with ultrasound field conformity, which can realize conformation coverage of the ultrasound field, further realize maximum utilization of ultrasound energy, and thus improve gene transfection efficiency.

In a first aspect, an ultrasonic field conformal gene transfection device is provided, comprising: an ultrasonic transducer for emitting ultrasonic waves; an ultrasonic field shaper for containing a complex of genes, cells and ultrasound responsive particles, the shape and size of the ultrasonic field shaper being determined from the sound field of the ultrasonic waves; and the focal length adapter is used for fixing the ultrasonic sound field conformal device at a preset position, and the preset position is determined according to the sound field position of the ultrasonic wave.

According to the gene transfection device provided by the embodiment of the application, the ultrasonic field shape-adapting device and the ultrasonic transducer are mutually adapted and used, and the ultrasonic field shape-adapting device and the ultrasonic transducer have a corresponding relation. The size and the shape of the ultrasonic field shape-adapting device are determined according to the sound field of the ultrasonic waves emitted by the ultrasonic transducer, the ultrasonic field shape-adapting device is further fixed at a preset position, the preset position is positioned in the sound field and is determined according to the sound field position of the ultrasonic waves, the ultrasonic field shape-adapting device and the ultrasonic field are in shape-adapting coverage with each other, further, the maximum utilization of ultrasonic energy can be realized, and therefore, the gene transfection efficiency is improved.

In addition, according to the gene transfection device provided by the embodiment of the application, the targeted transmission property and the transient reversible permeability can be improved. The ultrasonic irradiation has organ targeting property, so that the irradiation local gene transfection efficiency can be improved, ultrasonic irradiation of a target part based on certain power is realized, ultrasonic response particles are broken, and carried medicines or genes are released. The ultrasonic response particles are broken to generate cavitation effect, including sound hole effect, micro jet flow and the like, so that micro blood vessels or cell membranes and barriers in vivo generate instant reversible permeability increase, and the permeation of genes can be promoted. Therefore, the gene transfection device provided by the application further enables the gene transfection technology of the cells to realize high-efficiency transfection and universality, and has a very wide application prospect.

Alternatively, the ultrasound-responsive particles contained by the ultrasound field adapter may be nanoparticles or microparticles, such as biological nanobubbles and the like.

Alternatively, the ultrasound responsive particles may be ultrasound microbubbles.

For example, the ultrasound microbubble can be a lipid ultrasound microbubble, such as a phospholipid vesicle, a PLGA microbubble, or the like.

Alternatively, the cells accommodated by the ultrasonic field adapter can be any one of suspension cells, adherent cells, stem cells or primary cells and the like.

In one possible design, the shape of the ultrasonic sound field adapter is determined according to the shape of a focal spot of the sound field, the size of the ultrasonic sound field adapter is determined according to the size of the focal spot, and the preset position is determined according to the position of the focal spot.

In one possible design, the shape of the ultrasonic sound field shapers is the same as the shape of the focal spot.

In one possible design, the size of the ultrasonic sound field shapers is the same as the size of the focal spot.

In one possible design, the preset position is a central position of the focal spot.

In one possible design, one end of the focal length adapter is used for connecting with the ultrasonic field adapter, and the other end of the focal length adapter is used for connecting with the ultrasonic transducer, and when the ultrasonic field adapter, the focal length adapter and the ultrasonic transducer are connected together, the ultrasonic field adapter is located at the preset position.

That is to say, the focus adapter that this application embodiment provided can play the effect of focus location, and when ultrasonic field shape-fitting ware, focus adapter and ultrasonic transducer three were connected to together, ultrasonic field shape-fitting ware can be located this preset position automatically, consequently does not need the position of manual adjustment ultrasonic field shape-fitting ware, has simplified the operating procedure from this, has guaranteed the convenience and the high efficiency of gene transfection.

In one possible design, the ultrasonic field shapers and the focal length adapter form a unitary structure through a unitary molding process. Through above setting, relative displacement can not take place between ultrasonic field shape-conforming ware and the focus adapter the two, and when focus adapter was connected on ultrasonic transducer, ultrasonic field shape-conforming ware can be located preset position automatically. Because the ultrasonic field adapter and the focal length adapter do not need to be connected, and the ultrasonic field adapter and the focal length adapter are fixedly connected together, relative displacement does not occur, the positioning error caused by the connection of the ultrasonic field adapter and the focal length adapter is avoided, the operation steps are further simplified, and the positioning accuracy is improved.

In one possible design, the ultrasonic field shapers and the focal length adapter are integrally formed by 3D printing technology.

For example, it may be integrally formed of photosensitive resin, plastic, rubber, glass, metal, or the like by a 3D printing technique.

In one possible design, the ultrasonic field adapter is fixedly connected to the inside of the focal length adapter.

In one possible design, the focal length adapter includes a top wall, the ultrasonic sound field shape-adapting device is fixedly arranged on an inner wall surface of the top wall, a sample loading port is formed in the top wall, and the sample loading port is communicated with an opening of the ultrasonic sound field shape-adapting device.

In one possible design, the top wall is vented.

In a possible design, the middle part of the top wall forms a concave structure, the height of the inner surface of the concave structure is lower than that of the inner surface of the other part of the top wall, and the sample loading port is opened in the concave structure.

By arranging the concave structure, on one hand, the material can be fed conveniently, and the compound is prevented from flowing into the external environment; on the other hand, 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, so that when bubbles exist, the bubbles are extruded to other parts of the top wall, the propagation of ultrasonic waves cannot be influenced by the bubbles, the problem of energy attenuation is avoided, and the gene transfection efficiency is improved. In this case, the air bubbles can be removed without providing the sample loading port.

In one possible design, 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, the other end of the peripheral wall is provided with a connector, and the focal length adapter is detachably connected with the ultrasonic transducer through the connector. The focal length adapter is detachably connected with the ultrasonic transducer through the connector, so that the focal length adapter and the ultrasonic transducer can be flexibly and simply connected. Meanwhile, different ultrasonic transducers are convenient to replace and use, so that different experimental or therapeutic requirements are met.

In one possible design, the ultrasound transducer is a focused ultrasound probe.

In one possible design, the ultrasound responsive particles are ultrasound microbubbles.

In one possible design, each of the ultrasonic transducers and the ultrasonic sound field conformality device comprises a plurality of ultrasonic transducers, the plurality of ultrasonic transducers correspond to the plurality of ultrasonic sound field conformality devices in a one-to-one correspondence mode, and the sound field of ultrasonic waves emitted by each ultrasonic transducer is different.

Thus, when in use, the ultrasonic transducer can be firstly determined to be selected for operation according to experiments and medical requirements, and after the ultrasonic transducer is determined, an ultrasonic sound field adapter matched with the ultrasonic transducer can be further selected for operation.

Drawings

FIG. 1 is a schematic diagram showing the overall structure of a gene transfection apparatus provided in the examples of the present application.

FIG. 2 is an exploded view of a partial structure of a gene transfection apparatus provided in the examples of the present application.

Fig. 3 is a focal spot diagram measured using an ultrasonic acoustic beam analyzer provided by an embodiment of the present application.

FIG. 4 is a comparison of transfection under different experimental conditions by fluorescence microscopy.

Detailed Description

The technical solution in the present application will be described below with reference to the accompanying drawings. It is to be understood that the embodiments described are only a few embodiments of the present application and not all embodiments.

In the description of the present application, it is to be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; may be mechanically connected, may be electrically connected or may be in communication with each other; either directly or indirectly through intervening media, either internally or in any other relationship. The specific meaning of the above terms in the present application can be understood by those of ordinary skill in the art as appropriate.

In the description of the present application, it is to be understood that the terms "upper", "lower", "side", "inner", "outer", "top", "bottom", and the like, indicate orientations or positional relationships based on installation, are only used for convenience in describing the present application and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present application.

It should be noted that the same reference numerals are used to denote the same components or parts in the embodiments of the present application, and for the same parts in the embodiments of the present application, only one of the parts or parts may be given the reference numeral, and it should be understood that the reference numerals are also applicable to the other same parts or parts.

Gene therapy, as a new milestone in biotechnology, is one of the mainstream research directions in the development of biotechnology industry. Gene therapy is the treatment of disease by introducing foreign genes into target cells in a specific manner to correct defective genes or to produce corresponding biological effects. In recent years, with the development of molecular biology, gene therapy has shown unique advantages in the treatment of major diseases. Research shows that the three elements of gene therapy are target gene, gene transfection and target cell, and under certain conditions of the target cell, the gene transfection technology is the key for determining the gene therapy effect. Among them, the gene delivery system which is noninvasive, highly targeted, highly controllable, and capable of effectively expressing is of great importance in clinical practice. However, how to deliver genes to a target site safely and efficiently and to enable stable and persistent expression in tissue cells is a significant challenge and challenge for gene therapy. Therefore, the search for a new gene transfection method and device has become an urgent task for researchers.

Currently, vectors used for gene transfection can be divided into two major categories: viral vectors and non-viral vectors. Common virus vector systems comprise transcription viruses, lentiviruses, adenoviruses, adeno-associated viruses and the like, have high transfection efficiency, but have the problems of safety, autoimmunity, lack of tumor targeting specificity, small carrying capacity of target genes, difficulty in large-scale application and bottleneck of restricting further application of virus vectors. Non-viral vectors such as cationic liposome and the like provide a safer gene delivery method for people, and have the advantages of low immunogenicity, simple structure, easy chemical modification to change the functions, easy large-scale production and preparation and the like. However, the gene transfection efficiency using these non-viral vectors is low, and improving the gene transfection efficiency has been the target of the research of biomedical researchers. The safety advantage of non-viral vectors over most viral vectors is becoming increasingly appreciated by researchers.

Recent researches show that ultrasonic microvesicles are used as a carrier, and ultrasonic irradiation is carried out simultaneously to transfect a target gene in a targeted manner so as to generate a certain biological effect, namely an Ultrasonic Targeted Microvesicle Destruction (UTMD) mediated gene transfection method. The UTMD-mediated gene transfection method provides a new technology for clinical application of gene therapy, and avoids the defects of the conventional virus and non-virus vector systems. The principle is that the gas-containing ultrasonic microvesicles are continuously vibrated, expanded and contracted under the ultrasonic irradiation with certain sound intensity and mechanical index action to generate rupture, so that instant cavitation effect is generated to increase the permeability of cell membranes and generate temporary and reversible sound holes on the cell membranes, and genes combined with the microvesicles enter cells through the sound holes. UTMD has been applied to gene transfection studies of various animal experimental disease models to obtain safe transfection results.

However, the existing UTMD still has its application drawbacks. Specifically, existing UTMD ultrasonic transfectants generally use culture vessels such as petri dishes and plates to carry a complex of cells, genes, and ultrasonic microbubbles, and perform ultrasonic irradiation by a focused ultrasonic probe. The focused ultrasonic probe can focus an ultrasonic beam to a smaller area and has the characteristics of strong directivity, good penetrability, good aggregability, good energy precipitability and the like. However, the irradiation sound field is not uniformly distributed, the sound field can only cover a part of the culture container, and the cells are usually dispersed in each part of the culture container, that is, the ultrasonic irradiation can only cover a part of the cells in the culture container, thereby causing low energy utilization rate of the ultrasonic probe irradiation, and further causing low gene transfection efficiency.

Therefore, aiming at the defects, the application improves the existing ultrasonic-mediated gene transfection device, and provides the ultrasonic sound field conformal gene transfection device, which can realize conformal coverage on the ultrasonic sound field, further realize maximum utilization of ultrasonic energy, and further improve the gene transfection efficiency.

FIG. 1 is a schematic diagram showing the overall structure of a gene transfection apparatus provided in the examples of the present application. FIG. 2 is an exploded view of a partial structure of a gene transfection apparatus provided in the examples of the present application.

As shown in fig. 1 and 2, the gene transfection device provided in the embodiments of the present application can achieve conformal coverage of an ultrasonic field, and includes an ultrasonic transducer 1, an ultrasonic field shaper 2, and a focal length adapter 3.

Wherein the ultrasonic transducer 1 is used for emitting ultrasonic waves. The ultrasonic field shaper 2 is used to contain a complex of genes, cells and ultrasound responsive particles, and the shape and size of the ultrasonic field shaper 2 are determined according to the sound field of the ultrasonic waves. The focal length adaptor 3 is used to fix the ultrasonic sound field adapter 2 at a preset position determined according to the sound field position of the ultrasonic wave.

Specifically, the ultrasonic field former 2 containing the complex of the gene, the cell and the ultrasonic response particle is fixed at a preset position through the focal length adapter 3, and the ultrasonic transducer 1 can convert the electric signal into an ultrasonic signal and transmit the ultrasonic energy into the ultrasonic field former 2 to perform ultrasonic irradiation on the complex of the gene, the cell and the ultrasonic response particle. Under the action of the ultrasonic irradiation, the ultrasonic response particles are continuously vibrated, expanded and contracted to break, so that an instant cavitation effect is generated to increase the permeability of cell membranes and generate temporary and reversible sound holes on the cell membranes, and genes can enter cells through the sound holes to achieve the purpose of gene transfection.

According to the gene transfection device provided by the embodiment of the application, the ultrasonic field shaper 2 and the ultrasonic transducer 1 are mutually matched for use and have a corresponding relationship. The size and the shape of the ultrasonic field shape-adapting device 2 are determined according to the sound field of the ultrasonic wave emitted by the ultrasonic transducer 1, the ultrasonic field shape-adapting device 2 is further fixed at a preset position, the preset position is positioned in the sound field and is determined according to the sound field position of the ultrasonic wave, the ultrasonic field shape-adapting device 2 and the ultrasonic field are in shape-adapting coverage with each other, and then the maximum utilization of ultrasonic energy can be realized, so that the gene transfection efficiency is improved.

In the embodiment of the application, the ultrasonic response particles can respond to ultrasonic irradiation, and can vibrate, expand and contract under the action of ultrasonic waves to break, so that the instant cavitation effect is generated.

Alternatively, the ultrasound field adapter 2 may contain ultrasound-responsive particles that may be nanoparticles or microparticles, such as biological nanobubbles.

Alternatively, the cells accommodated in the ultrasonic field adapter 2 may be any one of suspension cells, adherent cells, stem cells, primary cells, or the like.

The ultrasonic transducer 1 is used to emit ultrasonic waves. Alternatively, the ultrasonic transducer 1 may be an ultrasonic probe, for example, a focused ultrasonic probe or a planar probe, etc.

Optionally, the frequency range of the ultrasonic wave emitted by the ultrasonic transducer 1 may be 0.5-10 MHz, such as 1-3 MHz, 1-5 MHz, 2-3 MHz, 3-7 MHz, 4-8 MHz, 5-10 MHz, and the like, and may also be 1MHz, 2MHz, 3MHz, 5MHz, 8MHz, 10MHz, and the like.

Optionally, the sound intensity range can be 0.1-5W/cm²For example, 0.25 to 3W/cm²，0.5～2W/cm²，3～5W/cm²Etc., and may be 1W/cm²，1.5W/cm²，2.0W/cm²，2.5W/cm²And the like.

Alternatively, the action time is 0.1 to 10 minutes, for example, 0.25 to 3 minutes, 2 to 5 minutes, 3 to 6 minutes, 5 to 8 minutes, etc., and further, 1 minute, 1.5 minutes, 4 minutes, 7 minutes, 9 minutes, etc., may be mentioned.

Optionally, the duty ratio may be 10-50%, for example, 15-45%, 20-40%, 15-30%, 30-50%, etc., and may be 20%, 25%, 35%, etc.

It should be understood that the above parameters are merely examples, and the ultrasound transducer 1 provided herein is not limited to other parameters.

As another example, the transducing material of the ultrasonic transducer 1 includes, but is not limited to, a magnetically compatible material.

The ultrasonic transducer 1 may 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, electric signals generated by the signal generator and the power amplifier are transmitted to the ultrasonic transducer 1 through a signal transmission line 11, and the electric signals are converted into ultrasonic waves on the ultrasonic transducer 1.

The degree of the sonoporation effect is related to factors such as sound pressure, sound intensity, frequency, work cycle and irradiation time of ultrasonic irradiation, and the working parameters of the ultrasonic signal output device 9 can be adjusted according to different experiments or medical requirements, so that energy safety can be realized, and the degree of the sonoporation effect can be adjusted and controlled.

The ultrasonic field shaper 2 is used to contain a complex of genes, cells and ultrasound responsive particles, and the shape and size of the ultrasonic field shaper 2 are determined according to the sound field of the ultrasonic waves. Alternatively, the shape and size of the ultrasonic sound field shapers 2 may be determined according to the shape, size, energy distribution and the like of the sound field, which is not limited in this application.

The ultrasonic sound field shapers 2 are further fixed at preset positions determined according to the sound field positions of the ultrasonic waves. Alternatively, the preset position may be determined according to the energy distribution of the sound field. For example, the predetermined position may be located on a central axis of the sound field, which is not limited in this application.

Fig. 3 is a focal spot diagram measured using an ultrasonic acoustic beam analyzer provided by an embodiment of the present application. In fig. 3, a place where two bright lines cross each other, that is, a region in a black elliptical circle, is a focal spot of the ultrasonic sound field. The focal spot is the region of the ultrasonic sound field where the energy density is the largest, and in the embodiment of the present application, the focal spot of the sound field may be determined first, and then the ultrasonic sound field shapers 2 may be set according to the focal spot. The application does not limit how the focal spot of the sound field is determined.

For example, the sound field of the ultrasonic transducer can be simulated by an acoustic detection instrument such as a hydrophone and an acoustic beam analyzer or by a computer according to the physical characteristics of the ultrasonic transducer, the size of the used focal spot is measured, the volume of the focal spot is calculated, and the central position of the focal spot is determined.

Alternatively, the shape of the ultrasonic sound field shapers 2 may be determined from the shape of the focal spot of the sound field. For example, the shape of the ultrasonic field shapers 2 may be the same as, or approximately the same as, the shape of the focal spot.

As shown in fig. 2, in the embodiment of the present application, the shape of the focal spot generated by the ultrasonic transducer 1 is an ellipsoid, and therefore the shape of the ultrasonic field shaper 2 is also set to be an ellipsoid.

In other embodiments, the shape of the focal spot may be other shapes such as a cylinder, a sphere, a solid polygon, and correspondingly, the shape of the ultrasonic field shaper 2 may also be set to be a cylinder, a sphere, a solid polygon, and the like.

It should be understood that the shape of the ultrasonic field shape-adapting device 2 and the shape of the focal spot may also be different, for example, the shape of the focal spot is spherical, and in this case, the shape of the ultrasonic field shape-adapting device 2 may be set to be an ellipsoid shape or a solid polygon shape, which is not limited in this application.

The size of the ultrasonic field shapers 2 can be determined according to the size of the focal spot. For example, the size of the ultrasonic field shapers 2 may be the same as or similar (approximately the same) as the size of the focal spot.

Alternatively, the size (or volume) of the ultrasonic sound field shapers 2 may be equal to, slightly larger than or slightly smaller than the size (or volume) of the focal spot.

Alternatively, the size of the ultrasonic field shaper 2 and the size of the focal spot may be proportional, for example, the size of the ultrasonic field shaper 2 may 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 ultrasound field shaper 2 in a preset position, which can be determined from the position of the focal spot.

Alternatively, the preset position may be determined from the center position of the focal spot. For example, the preset position may be adjacent to a central position of the focal spot, or coincide with a central position of the focal spot.

That is, the center of the ultrasonic field shaper 2 may be disposed adjacent to the center of the focal spot, or the center of the ultrasonic field shaper 2 and the center of the focal spot may coincide with each other.

In the embodiment of the present application, the shape of the ultrasonic field shaper 2 and the shape of the focal spot are the same, and the size is the same, and the center position of the ultrasonic field shaper 2 and the center position of the focal spot coincide with each other. Through above setting, can maximize the utilization ultrasonic energy, and then can improve transfection efficiency.

In the embodiment of the present application, the ultrasonic field adapter 2 is fixed at a preset position by the focal length adapter 3, and the specific form of the focal length adapter 3 is not limited in the present application. In the present embodiment, the focal length adapter 3 is a collimator.

Alternatively, in other embodiments, the focal length adaptor 3 may have other structures, for example, the focal length adaptor 3 may be any one of a fixed bracket, a wire, a pull rope, and the like.

As shown in fig. 1 and 2, in the embodiment of the present application, one end of the focal length adapter 3 is used to connect with the ultrasonic field shaper 2, and the other end of the focal length adapter 3 is used to connect with the ultrasonic transducer 1, and when the ultrasonic field shaper 2 and the focal length adapter 3 are connected together with the ultrasonic transducer 1, the ultrasonic field shaper 2 is located at the preset position.

That is to say, the focus adapter 3 that this application embodiment provided can play the effect of focus location, and when ultrasonic field shape-fitting ware 2, focus adapter 3 and ultrasonic transducer 1 three were connected to together, ultrasonic field shape-fitting ware 2 can be located this preset position automatically, consequently does not need the position of manual adjustment ultrasonic field shape-fitting ware 2, has simplified operating procedure from this, has guaranteed the convenience and the high efficiency of gene transfection.

In the embodiment of the present application, the ultrasonic field shaper 2 is fixedly connected to the focal length adaptor 3, and is formed into an integral structure through an integral molding process.

Through the above arrangement, relative displacement does not occur between the ultrasonic field shapers 2 and the focal length adapter 3, and when the focal length adapter 3 is connected above the ultrasonic transducer 1, the ultrasonic field shapers 2 can be automatically located at the preset position. Because the ultrasonic field adapter 2 and the focal length adapter 3 do not need to be connected, and the ultrasonic field adapter 2 and the focal length adapter are fixedly connected together, relative displacement does not occur, and a positioning error caused by the connection of the ultrasonic field adapter and the focal length adapter is avoided, so that the operation steps are further simplified, and the positioning accuracy is improved.

For example, the integral molding process may be injection molding.

For another example, the ultrasonic field shaper 2 and the focal length adaptor 3 may also be integrally formed by 3D printing technology. At this time, the two may be integrally formed by a 3D printing technique from a material such as photosensitive resin, plastic, rubber, glass, metal, or the like.

Alternatively, in other embodiments, the ultrasonic field adapter 2 and the focal length adapter 3 may also be made of other materials such as glass, metal, plastic, and the like, which is not limited in this application.

Alternatively, in other embodiments, the ultrasonic field shapers 2 and the focal length adapter 3 may be detachably connected.

As shown in fig. 2, in the present embodiment, 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 disposed around the circumference of the top wall 32, the other end of the peripheral wall 31 is provided with a connector 7, and the focal length adapter 3 is detachably connected to the ultrasonic transducer 1 through the connector 7.

The focal length adapter 3 is detachably connected with the ultrasonic transducer 1 through the connector 7, so that flexible and simple connection between the focal length adapter and the ultrasonic transducer can be realized. Meanwhile, different ultrasonic transducers are convenient to replace and use, so that different experimental or therapeutic requirements are met.

In this embodiment, the connector 7 may be a connection port between the ultrasonic transducer 1 and the focal length adapter 3, and is designed as a sliding groove, disposed on an inner wall surface of a lower end of the focal length adapter 3, and capable of being fixedly connected to an upper end of the ultrasonic transducer 1. In other embodiments, the connector 7 may also be another structure capable of detachably connecting the ultrasound transducer 1 and the focal length adapter 3, which is not limited in this application.

As shown in fig. 2, the ultrasonic field shaper 2 is fixedly attached to the inside of the focal length adapter 3. Specifically, the ultrasonic field shape-adapting device 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 is communicated with the opening of the ultrasonic field shape-adapting device 2. Thereby enabling the delivery of a complex of genes, cells and ultrasound responsive particles into the ultrasound field shapers 2.

At this time, since the ultrasonic field adapter 2 is not sealed, the loading port 4 may be sealed with a sealing film during use in order to avoid contamination of cells.

In other embodiments, the focal length adapter 3 may also be fixedly disposed on the peripheral wall 31, and the sample loading port 4 is correspondingly opened on the peripheral wall 31.

In another embodiment, the sample loading port 4 may not be provided in the focal length adapter 3, and the ultrasonic field adapter 2 may be fixed to the inside of the focal length adapter 3 after the compound is injected into the ultrasonic field adapter 2.

Further, as shown in FIGS. 1 and 2,

the middle part of the top wall 32 forms a concave structure 6, the height of the inner surface of the concave structure 6 is lower than that of the inner surface of the other part of the top wall 32, and the sample loading port 4 is arranged in the concave structure 6.

By arranging the concave structure 6, on one hand, the feeding can be facilitated, and the compound is prevented from flowing into the external environment; on the other hand, the height of the inner surface of the concave structure 6 is lower than that of the inner surface of the other part of the top wall 32, so that when bubbles exist, the bubbles are extruded to the other part of the top wall 32, the propagation of ultrasonic waves is not affected by the bubbles, the problem of energy attenuation is avoided, and the gene transfection efficiency is improved. In this case, the air bubbles can be removed without providing the sample application port 4.

Further, in order to conduct the ultrasonic wave, the focus adapter 3 may be filled with a conducting medium, which may be any one of water, coupling liquid, PVA, or the like.

Further, in order to prevent the ultrasonic wave propagation from being affected by the air bubbles and to avoid energy attenuation, an air vent 5 may be formed in the top wall 32, and the air in the focal length adapter 3 may be exhausted through the air vent 5.

For example, before use, the inside of the focus adapter 3 should be checked for the presence of air bubbles, and if there are air bubbles, air should be discharged through the air discharge hole 5. The degassing method is to inject the medium (most commonly water) using a 50ml syringe until the bubbles disappear, and ultrasonic irradiation can be performed.

As shown in fig. 1, the gene transfection apparatus provided in the embodiment of the present application further includes a holder 8. The holder 8 is used for fixing the ultrasonic transducer 1, the ultrasonic sound field adapter 2, the focal length adapter 3 and other components.

Alternatively, the holder 8 may be a gantry, and the ultrasonic transducer 1, the ultrasonic sound field shaper 2, and the focal length adaptor 3 are fixed thereon by the gantry.

Optionally, the center of the holder 8 may be provided with a small hole to facilitate the connection of the signal transmission line 11 with the ultrasonic transducer 1.

As shown in fig. 1, the gene transfection apparatus provided in the embodiment of the present application further includes a medium container 10, and the medium container 10 contains an ultrasonic propagation medium such as water.

In order to meet different experimental and medical requirements, in the embodiment of the present application, the ultrasonic transducers 1 and the ultrasonic field shapers 2 each include a plurality of ultrasonic transducers 1, the plurality of ultrasonic transducers 1 correspond to the plurality of ultrasonic field shapers 2 one to one, and the sound fields of the ultrasonic waves emitted by the respective ultrasonic transducers 1 are different from each other.

Thus, when in use, it can be firstly determined which ultrasonic transducer 1 is selected to operate according to experiments and medical requirements, and after the ultrasonic transducer 1 is determined, an ultrasonic sound field adapter 2 matched with the ultrasonic transducer 1 can be further selected to operate.

Alternatively, when the ultrasonic field shapers 2 and the focal length adapters 3 are of an integral structure, after the ultrasonic transducer 1 is determined, the focal length adapter 3 matched with the ultrasonic field shapers can be further selected to work.

The research team of the inventor utilizes the gene transfection device provided by the embodiment of the application to carry out experimental verification, and utilizes the sonoporation effect to realize the high-efficiency gene transfection of 293T cells. The experimental steps are as follows:

1. the gene transfection apparatus is prepared in advance, the holder 8 is placed in water in the medium container 10, the ultrasonic transducer 1 is placed thereon, and the corresponding focus adapter 3 is fitted, and air bubbles are removed through the air vent 5. Starting the ultrasonic signal output device 9, setting the transfection parameters of the ultrasonic signal output device to be 1-3 Mhz frequency, 10-50% duty ratio and 0.1-10 min irradiation time.

2. Calculating the number of prepared phospholipid vesicles by using a particle counting analyzer;

3. each hole takes 5 x 10⁵-5*10⁸Mixing phospholipid vesicle and 0.5-5ug plasmid, making into transfected compound, standing at room temperature for 10-30 min;

4. the cells were digested and counted at 10-20 x 10 per well⁴Adding the prepared compound of phospholipid vesicles and plasmids into cells, placing the compound into an ultrasonic sound field shape-adapting device 2 by arranging a sample loading port 4 on a focal length adapter 3, sealing the sample loading port 4 by a sealing film, and then irradiating according to the ultrasonic parameters of experimental design, and paying attention to aseptic operation.

5. After the operation is finished, the cells are recovered from the ultrasonic field shape-adapting device 2, DMEM culture medium is added as soon as possible, the DMEM culture medium is paved on a 24-hole plate, incubation is carried out for 24 hours, and the transfection condition is observed by a fluorescence microscope after 24 hours. Finally, the results of this experiment are shown in fig. 4 (c).

In order to clearly understand the performance of the gene transfection device provided in the examples of the present application, the research team of the inventors also performed comparative experiments, and fig. 4 is a comparative graph of transfection conditions observed by a fluorescence microscope under different experimental conditions.

In fig. 4(a), the blank control group, cells alone, and no complex of phospholipid vesicles and genes were added, and as a result, no cells expressed mCherry fluorescent protein. FIG. 4(b) shows the phospholipid vesicle + gene complex, but with the ultrasound irradiation using the prior art gene transfection device, very few cells expressed fluorescent protein; 4(c) is a phospholipid vesicle + gene-added compound, and ultrasonic irradiation is carried out by using the gene transfection device provided by the embodiment of the application, so that most cells express fluorescent protein. Experimental results prove that the gene transfection device provided by the embodiment of the application can improve the gene transportation effect and has higher transfection efficiency.

The above description is only for the specific embodiments of the present application, but the scope of the present application is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present application, and shall be covered by the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims

1. An ultrasonic field conformable gene transfection device, comprising:

an ultrasonic transducer (1) for emitting ultrasonic waves;

an ultrasonic field shaper (2) for containing a complex of genes, cells and ultrasound responsive particles, the shape and size of the ultrasonic field shaper (2) being determined from the sound field of the ultrasonic waves;

the focal length adapter (3) is used for fixing the ultrasonic sound field adapter (2) at a preset position, and the preset position is determined according to the sound field position of the ultrasonic wave.

2. The gene transfection device according to claim 1, characterized in that the shape of the ultrasonic acoustic field shaper (2) is determined according to the shape of a focal spot of the acoustic field, the size of the ultrasonic acoustic field shaper (2) is determined according to the size of the focal spot, and the preset position is determined according to the position of the focal spot.

3. Gene transfection device according to claim 2, characterized in that the shape of the ultrasonic acoustic field shaper (2) is the same as the shape of the focal spot.

4. Gene transfection device according to claim 2 or 3, characterized in that the size of the ultrasonic field shaper (2) is the same as the size of the focal spot.

5. A gene transfection device according to claim 2 or 3 wherein the predetermined position is a central position of the focal spot.

6. Gene transfection device according to any one of claims 1 to 3, characterized in that one end of the focal length adapter (3) is adapted to be connected to the ultrasonic field adapter (2) and the other end of the focal length adapter (3) is adapted to be connected to the ultrasonic transducer (1), the ultrasonic field adapter (2) being located at the predetermined position when the ultrasonic field adapter (2), the focal length adapter (3) and the ultrasonic transducer (1) are connected together.

7. The gene transfection device according to claim 6, characterized in that the ultrasonic field shaper (2) and the focal length adapter (3) are formed as a unitary structure by a unitary molding process.

8. Gene transfection device according to claim 7, characterized in that the ultrasonic field shaper (2) is made in one piece with the focal length adapter (3) by 3D printing technology.

9. Gene transfection device according to claim 6, characterized in that the ultrasonic field shaper (2) is fixedly connected to the inside of the focal length adapter (3).

10. The gene transfection device according to claim 9, wherein the focal length adapter (3) comprises a top wall (32), the ultrasonic field shape-adapting device (2) is fixedly arranged on the inner wall surface of the top wall (32), a sample loading port (4) is formed in the top wall (32), and the sample loading port (4) is communicated with an opening of the ultrasonic field shape-adapting device (2).

11. A gene transfection device according to claim 10, characterized in that the top wall (32) is provided with vent holes (5).

12. Gene transfection device according to claim 10 or 11, characterized in that the middle of the top wall (32) forms a concave structure (6), the height of the inner surface of the concave structure (6) is lower than the height of the inner surface of the rest of the top wall (32), and the sample loading port (4) opens on the concave structure (6).

13. Gene transfection device according to claim 10 or 11, characterized in that the focal length adapter (3) further comprises a circumferential wall (31), one end of the circumferential wall (31) is connected to the top wall (32) and arranged around the circumference of the top wall (32), the other end of the circumferential wall (31) is provided with a connector (7), and the focal length adapter (3) is detachably connected with the ultrasound transducer (1) by means of the connector (7).

14. A gene transfection device according to any one of claims 1 to 3, characterized in that the ultrasonic transducer (1) is a focused ultrasonic probe.

15. A gene transfection device according to any one of claims 1 to 3 wherein the ultrasound responsive particles are ultrasound microbubbles.

16. The gene transfection device according to any one of claims 1 to 3, wherein the ultrasonic transducers (1) and the ultrasonic field shapers (2) each comprise a plurality of ultrasonic transducers (1) and a plurality of ultrasonic field shapers (2), the plurality of ultrasonic transducers (1) correspond to the plurality of ultrasonic field shapers (2) one by one, and the sound fields of the ultrasonic waves emitted by the respective ultrasonic transducers (1) are different from each other.