A gene transfection device with conformable ultrasonic sound field

Abstract

The present application provides a gene transfection device with conformal ultrasonic field, comprising: an ultrasonic transducer (1) for emitting ultrasonic waves; a conformal ultrasonic field device (2) for accommodating a complex of genes, cells and ultrasonic response particles, wherein the shape and size of the conformal ultrasonic field device (2) are determined according to the ultrasonic field of the ultrasonic waves; and a focal length adapter (3) for fixing the conformal ultrasonic field device (2) at a preset position, wherein the preset position is determined according to the position of the ultrasonic field of the ultrasonic waves. The gene transfection device provided by the present application can achieve conformal coverage of the ultrasonic field, thereby realizing the maximum utilization of ultrasonic energy, and thus improving the gene transfection efficiency.

Description

Technical Field

[0001] This application relates to the field of scientific research devices used in biomedical experiments, and particularly to a gene transfection device with conformal ultrasound field. Background Art

[0002] Gene therapy aims to treat diseases by introducing normal genes or therapeutic genetic materials into target cells in a specific manner to correct gene defects or exert therapeutic effects. The three elements of gene therapy are the target gene, gene transfection, and target cells. Given a certain target cell, gene transfection technology is the key to determining the efficacy of gene therapy. Among them, a non-invasive, highly targeted, highly controllable, and effectively expressible gene delivery system is particularly important clinically. However, how to safely and efficiently deliver genes to the targeted site and enable stable and persistent expression in tissue cells is a major problem and challenge faced by gene therapy.

[0003] In recent years, the gene transfection method mediated by ultrasound targeted microbubble destruction (UTMD) has provided a new technology for the clinical application of gene therapy. Its principle is that gas-containing ultrasound microbubbles vibrate, expand, contract, and rupture under the irradiation of ultrasound with a certain sound intensity and mechanical index, thereby generating an instantaneous cavitation effect that increases the cell membrane permeability and produces temporary and reversible sonopores on the cell membrane, enabling the genes bound to the microbubbles to enter the cells through the sonopores. As an ideal gene transfection method, UTMD has broad application prospects.

[0004] Existing UTMD still has its application defects. Specifically, existing UTMD ultrasound transfectors usually use culture containers such as petri dishes and culture plates to carry the complexes of cells, genes, and ultrasound microbubbles, and perform ultrasound irradiation through a focused ultrasound probe. However, the sound field distribution irradiated by the probe is uneven, and the sound field can only cover a part of the culture container, while the cells are usually dispersed in various parts of the culture container, resulting in low utilization efficiency of the irradiation energy of the ultrasound probe and thus low gene transfection efficiency. Summary of the Invention

[0005] This application provides a gene transfection device with conformal ultrasound field, which can achieve conformal coverage of the ultrasound field, thereby maximizing the utilization of ultrasound energy and improving the gene transfection efficiency.

[0006] In a first aspect, a gene transfection device with conformable ultrasonic sound field is provided, including: an ultrasonic transducer for emitting ultrasonic waves; a conformable ultrasonic sound field device for accommodating a complex of genes, cells and ultrasonic response particles, the shape and size of the conformable ultrasonic sound field device being determined according to the sound field of the ultrasonic waves; and a focal length adapter for fixing the conformable ultrasonic sound field device at a preset position, the preset position being determined according to the position of the sound field of the ultrasonic waves.

[0007] According to the gene transfection device provided by the embodiments of the present application, the conformable ultrasonic sound field device and the ultrasonic transducer are used in mutual adaptation and have a corresponding relationship. The size and shape of the conformable ultrasonic sound field device are determined according to the sound field of the ultrasonic waves emitted by the ultrasonic transducer. The conformable ultrasonic sound field device is further fixed at a preset position, which is located within the sound field and is determined according to the position of the sound field of the ultrasonic waves. The conformable ultrasonic sound field device and the ultrasonic sound field conform to and cover each other, thereby enabling the maximization of the utilization of ultrasonic energy and improving the gene transfection efficiency.

[0008] In addition, according to the gene transfection device provided by the embodiments of the present application, the target delivery characteristics and instantaneous reversible permeability can be improved. The ultrasonic irradiation itself has organ targeting, which can improve the gene transfection efficiency at the irradiated local part. Based on the ultrasonic irradiation with a certain power, the ultrasonic response particles rupture and release the carried drugs or genes. The rupture of the ultrasonic response particles generates cavitation effects, including sonoporation, micro-jet, etc., which cause the instantaneous reversible increase in the permeability of microvessels, cell membranes and in vivo barriers, and can promote the penetration of genes. Therefore, the gene transfection device provided by the present application further enables the gene transfection technology of cells to achieve efficient transfection and universality, and has a very broad application prospect.

[0009] Optionally, the ultrasonic response particles accommodated in the conformable ultrasonic sound field device can be nanoparticles or microparticles, such as biological nanobubbles, etc.

[0010] Optionally, the ultrasonic response particles can be ultrasonic microbubbles.

[0011] For example, the ultrasonic microbubbles can be lipid ultrasonic microbubbles, such as phospholipid vesicles, PLGA microbubbles, etc.

[0012] Optionally, the cells accommodated in the conformable ultrasonic sound field device can be any one of suspension cells, adherent cells, stem cells or primary cells, etc.

[0013] In a possible design, the shape of the conformable ultrasonic sound field device is determined according to the shape of the focal spot of the sound field, the size of the conformable ultrasonic sound field device is determined according to the size of the focal spot, and the preset position is determined according to the position of the focal spot.

[0014] In a possible design, the shape of the ultrasonic sound field conformer is the same as the shape of the focal spot.

[0015] In a possible design, the size of the ultrasonic sound field conformer is the same as the size of the focal spot.

[0016] In a possible design, the preset position is the central position of the focal spot.

[0017] In a possible design, one end of the focal length adapter is used to connect to the ultrasonic sound field conformer, and the other end of the focal length adapter is used to connect to the ultrasonic transducer. When the ultrasonic sound field conformer, the focal length adapter, and the ultrasonic transducer are connected together, the ultrasonic sound field conformer is located at the preset position.

[0018] That is to say, the focal length adapter provided in the embodiments of the present application can play a role in focusing and positioning. When the ultrasonic sound field conformer, the focal length adapter, and the ultrasonic transducer are connected together, the ultrasonic sound field conformer can automatically be located at the preset position. Therefore, it is not necessary to manually adjust the position of the ultrasonic sound field conformer, thereby simplifying the operation steps and ensuring the convenience and high efficiency of gene transfection.

[0019] In a possible design, the ultrasonic sound field conformer and the focal length adapter are formed into an integral structure by an integral molding process. With the above settings, there will be no relative displacement between the ultrasonic sound field conformer and the focal length adapter. When the focal length adapter is connected to the ultrasonic transducer, the ultrasonic sound field conformer can automatically be located at the preset position. Since there is no need to perform a connection operation on the ultrasonic sound field conformer and the focal length adapter, and the two are fixedly connected together without relative displacement, the positioning error caused by the connection of the above two is avoided, thereby further simplifying the operation steps and improving the positioning accuracy.

[0020] In a possible design, the ultrasonic sound field conformer and the focal length adapter are integrally formed by 3D printing technology.

[0021] For example, it can be integrally formed by 3D printing technology with photosensitive resin, plastic, rubber, glass, metal, etc.

[0022] In a possible design, the ultrasonic sound field conformer is fixedly connected inside the focal length adapter.

[0023] In a possible design, 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, and a sample loading port is opened on the top wall, and the sample loading port is communicated with the opening of the ultrasonic sound field conformer.

[0024] In a possible design, exhaust holes are formed in the top wall.

[0025] In a possible design, 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 that of the inner surfaces of other parts of the top wall, and the sample loading port is arranged in the concave structure.

[0026] By providing the concave structure, on the one hand, it is convenient to load materials and prevent the composite from flowing into the external environment; on the other hand, the height of the inner surface of the concave structure is lower than that of the inner surfaces of other parts of the top wall. In this way, when there are bubbles, the bubbles will be displaced to other parts of the top wall, so that the propagation of ultrasonic waves will not be affected by the bubbles, avoiding the problem of energy attenuation, and thus being beneficial to improving the efficiency of gene transfection. At this time, even if the sample loading port is not provided, it can also play a role in removing bubbles.

[0027] In a possible design, the focal length adapter further includes a peripheral wall, one end of the peripheral wall is connected to the top wall and is arranged around the circumference of the top wall, and a connector is arranged at the other end of the peripheral wall. The focal length adapter is detachably connected to the ultrasonic transducer through the connector. The detachable connection of the focal length adapter to the ultrasonic transducer through the connector can achieve a flexible and simple connection between the two. At the same time, it is also convenient to replace and use different ultrasonic transducers to meet the needs of different experiments or treatments.

[0028] In a possible design, the ultrasonic transducer is a focused ultrasonic probe.

[0029] In a possible design, the ultrasonic response particles are ultrasonic microbubbles.

[0030] In a possible design, both the ultrasonic transducer and the ultrasonic sound field conformer include a plurality of them. The plurality of ultrasonic transducers correspond to the plurality of ultrasonic sound field conformers one by one, and the sound fields of the ultrasonic waves emitted by each ultrasonic transducer are different.

[0031] In this way, during use, it is possible to first determine which ultrasonic transducer to select for operation according to the experimental and medical needs. After determining the ultrasonic transducer, it is possible to further select a matching ultrasonic sound field conformer for operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a schematic diagram of the overall structure of the gene transfection device provided by an embodiment of the present application.

[0033] Figure 2 is an exploded schematic diagram of a partial structure of the gene transfection device provided by an embodiment of the present application.

[0034] Figure 3It is the focal spot pattern measured by the ultrasonic beam analyzer provided by the embodiment of the present application.

[0035] Figure 4 It is a comparison chart of the transfection situation observed by a fluorescence microscope under different experimental conditions. Specific embodiments

[0036] Next, the technical solutions in the present application will be described in conjunction with the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments.

[0037] In the description of the present application, it should be noted that unless otherwise clearly specified and limited, the terms "installation", "connection", and "connection" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, an electrical connection, or a connection that can communicate with each other; it can be directly connected, or indirectly connected through an intermediate medium, and it can be the internal communication of two components or the interaction relationship between two components. For those of ordinary skill in the art, the specific meanings of the above terms in the present application can be understood according to specific situations.

[0038] In the description of the present application, it should be understood that the orientation or positional relationship indicated by the terms "upper", "lower", "side", "inner", "outer", "top", "bottom", etc. is based on the installed orientation or positional relationship, and is only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or component referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present application.

[0039] It should also be noted that in the embodiments of the present application, the same reference numerals are used to represent the same component or the same part. For the same parts in the embodiments of the present application, only one of the parts or components may be marked with a reference numeral in the figure. It should be understood that for other identical parts or components, the reference numerals are equally applicable.

[0040] As a new milestone in biotechnology, gene therapy is one of the mainstream research directions in the development of the biotechnology industry. Gene therapy is to introduce exogenous 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. 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 the target gene, gene transfection, and target cells. Under the condition of certain target cells, gene transfection technology is the key to determining the effect of gene therapy. Among them, a gene delivery system that is non-invasive, highly targeted, highly controllable, and can be effectively expressed is particularly important clinically. However, how to safely and efficiently deliver genes to the targeted site and stably and persistently express them in tissue cells is a major problem and challenge faced by gene therapy. Therefore, exploring a new gene transfection method and transfection device has become an urgent task for researchers.

[0041] At present, the vectors used for gene transfection can be divided into two categories: viral vectors and non-viral vectors. Common viral vector systems include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, etc. The transfection efficiency is relatively high, but their safety issues, autoimmunogenicity, lack of tumor targeting specificity, small capacity for carrying target genes, and difficulty in large-scale application are still the bottlenecks restricting the further application of viral vectors. Non-viral vectors, such as cationic liposomes, etc., provide us with a safer gene transport method, with advantages such as low immunogenicity, simple structure, easy chemical modification to change their functions, and easy large-scale production and preparation. However, the gene transfection efficiency using these non-viral vectors is relatively low. Improving gene transfection efficiency has always been the goal explored by biomedical researchers. Compared with most viral vectors, non-viral vectors are increasingly valued by scholars for their safety advantages.

[0042] Recent studies have shown that using ultrasound microbubbles as carriers and simultaneously performing ultrasound irradiation can target the transfection of target genes and produce certain biological effects, which is called gene transfection mediated by ultrasound targeted microbubble destruction (UTMD). The gene transfection method mediated by UTMD provides a new technology for the clinical application of gene therapy, avoiding the defects of previous viral and non-viral vector systems. Its principle is to use gas-containing ultrasound microbubbles. Under the action of ultrasound irradiation with a certain sound intensity and mechanical index, they continuously vibrate, expand, contract, and rupture, thereby generating an instantaneous cavitation effect, which causes an increase in cell membrane permeability and the formation of temporary and reversible sonopores on the cell membrane. Thus, the genes bound to the microbubbles enter the cells through the sonopores. This theory of ultrasound cavitation effect and sonopore effect is the main mechanism for UTMD to promote gene transfection. At present, there have been gene transfection studies applying UTMD to various animal experimental disease models, and safe transfection results have been obtained.

[0043] However, existing UTMD still has its application defects. Specifically, existing UTMD ultrasonic transfectors usually use culture vessels such as petri dishes and culture plates to carry the complexes of cells, genes, and ultrasonic microbubbles, and perform ultrasonic irradiation through a focused ultrasonic probe. A focused ultrasonic probe is capable of focusing an ultrasonic beam onto a smaller area, featuring strong directivity, good penetrability, good aggregability, and good energy deposition. However, the sound field distribution irradiated by it is uneven, and the sound field can only cover a part of the culture vessel, while cells are usually dispersed in various parts of the culture vessel. That is to say, ultrasonic irradiation can only cover a part of the cells in the culture vessel, resulting in low utilization rate of the irradiation energy of the ultrasonic probe, and thus low gene transfection efficiency.

[0044] Therefore, aiming at the above defects, this application improves the existing ultrasonic-mediated gene transfection device, and proposes a gene transfection device with a conformable ultrasonic sound field, which can achieve conformable coverage of the ultrasonic sound field, and then achieve the maximum utilization of ultrasonic energy, thereby improving the gene transfection efficiency.

[0045] Figure 1 It is a schematic diagram of the overall structure of the gene transfection device provided by an embodiment of this application. Figure 2 It is an exploded schematic diagram of a part of the structure of the gene transfection device provided by an embodiment of this application.

[0046] As Figure 1 、 2 shown, the gene transfection device provided by an embodiment of this application can achieve conformable coverage of the ultrasonic sound field. The gene transfection device includes an ultrasonic transducer 1, an ultrasonic sound field conformer 2, and a focal length adapter 3.

[0047] Among them, the ultrasonic transducer 1 is used to emit ultrasonic waves. The ultrasonic sound field conformer 2 is used to accommodate the complexes of genes, cells, and ultrasonic response particles. The shape and size of the ultrasonic sound field conformer 2 are determined according to the ultrasonic sound field. 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 position of the ultrasonic sound field.

[0048] Specifically, the ultrasonic sound field conformer 2 that accommodates the complexes of genes, cells, and ultrasonic response particles is fixed at a preset position through the focal length adapter 3. The ultrasonic transducer 1 can convert an electrical signal into an ultrasonic signal and transmit the ultrasonic energy into the ultrasonic sound field conformer 2 to perform ultrasonic irradiation on the complexes of genes, cells, and ultrasonic response particles. Under this ultrasonic irradiation, the ultrasonic response particles vibrate, expand, contract, and rupture continuously, generating an instantaneous cavitation effect, which causes an increase in the permeability of the cell membrane and the formation of temporary and reversible sonopores on the cell membrane. Thus, genes can enter the cells through the sonopores to achieve the purpose of gene transfection.

[0049] According to the gene transfection device provided by the embodiments of the present application, the ultrasonic sound field conformer 2 and the ultrasonic transducer 1 are used in mutual adaptation, and there is a corresponding relationship between the two. 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. The ultrasonic sound field conformer 2 is further fixed at a preset position, which is located within the sound field and is determined according to the position of the sound field of the ultrasonic waves. The ultrasonic sound field conformer 2 and the ultrasonic sound field are conformally covered with each other, thereby enabling the maximum utilization of ultrasonic energy, and thus improving the gene transfection efficiency.

[0050] In addition, according to the gene transfection device provided by the embodiments of the present application, the targeted transmission characteristics and instantaneous reversible permeability can be improved. The ultrasonic irradiation itself has organ targeting, which can improve the gene transfection efficiency at the irradiated local part. Based on the ultrasonic irradiation with a certain power, the ultrasonic-responsive particles rupture and release the carried drugs or genes. The rupture of the ultrasonic-responsive particles generates a cavitation effect, including sonoporation, microjet, etc., which causes the microvessels, cell membranes, and in vivo barriers to have an instantaneous reversible increase in permeability, and can promote the penetration of genes. Therefore, the gene transfection device provided by the present application further enables the gene transfection technology of cells to achieve efficient transfection and universality, and has a very broad application prospect.

[0051] In the embodiments of the present application, the ultrasonic-responsive particles can respond to ultrasonic irradiation and can vibrate, expand, contract, and rupture under the action of ultrasonic waves, thereby generating an instantaneous cavitation effect.

[0052] Optionally, the ultrasonic-responsive particles accommodated in the ultrasonic sound field conformer 2 can be nanoparticles or microparticles, such as biological nanobubbles, etc.

[0053] Optionally, the ultrasonic-responsive particles can be ultrasonic microbubbles.

[0054] For example, the ultrasonic microbubbles can be lipid ultrasonic microbubbles, such as phospholipid vesicles, PLGA microbubbles, etc.

[0055] Optionally, the cells accommodated in the ultrasonic sound field conformer 2 can be any one of suspension cells, adherent cells, stem cells, or primary cells, etc.

[0056] The ultrasonic transducer 1 is used to emit ultrasonic waves. Optionally, the ultrasonic transducer 1 can be an ultrasonic probe, such as a focused ultrasonic probe or a planar probe, etc.

[0057] Optionally, the ultrasonic frequency range emitted by the ultrasonic transducer 1 can be 0.5 to 10 MHz, such as 1 to 3 MHz, 1 to 5 MHz, 2 to 3 MHz, 3 to 7 MHz, 4 to 8 MHz, 5 to 10 MHz, etc. In addition, it can also be 1 MHz, 2 MHz, 3 MHz, 5 MHz, 8 MHz, 10 MHz, etc.

[0058] Optionally, the sound intensity range can be 0.1 to 5 W / cm 2 , such as 0.25 to 3 W / cm 2 , 0.5 to 2 W / cm 2 , 3 to 5 W / cm 2 etc. In addition, it can also be 1 W / cm 2 , 1.5 W / cm 2 , 2.0 W / cm 2 , 2.5 W / cm 2 etc.

[0059] Optionally, 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.

[0060] Optionally, the duty cycle can be 10 to 50%, for example, 15 to 45%, 20 to 40%, 15 to 30%, 30 to 50%, etc. In addition, it can also be 20%, 25%, 35%, etc.

[0061] It should be understood that the above parameters are only examples, and the ultrasonic transducer 1 provided in the present application is not limited to other parameters.

[0062] For another example, the transducer material of the ultrasonic transducer 1 includes but is not limited to magnetically compatible materials.

[0063] The ultrasonic transducer 1 can be electrically connected to the ultrasonic signal output device 9 through the 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 signals generated by the signal generator and the power amplifier are transmitted to the ultrasonic transducer 1 through the signal transmission line 11, and the electrical signals are converted into ultrasonic waves on the ultrasonic transducer 1.

[0064] The degree of the acoustic pore effect is related to factors such as the sound pressure, sound intensity, frequency, duty cycle, irradiation time, etc. of the ultrasonic irradiation. According to different experimental or medical needs, the working parameters of the ultrasonic signal output device 9 can be adjusted, and thus energy safety and adjustable control can be achieved.

[0065] The ultrasonic sound field conformer 2 is used to accommodate the complexes of genes, cells and ultrasonic response particles. The shape and size of the ultrasonic sound field conformer 2 are determined according to the ultrasonic sound field. Optionally, the shape and size of the ultrasonic sound field conformer 2 can be determined according to the shape, size, energy distribution, etc. of the sound field. This application does not limit this.

[0066] The ultrasonic sound field conformer 2 is further fixed at a preset position, which is determined according to the position of the ultrasonic sound field. Optionally, the preset position can be determined according to the energy distribution of the sound field. For example, the preset position can be located on the central axis of the sound field. This application does not limit this.

[0067] Figure 3 is the focal spot pattern measured by using an ultrasonic beam analyzer provided in the embodiment of this application. In Figure 3 , where the two bright lines intersect, that is, the area within the black ellipse, 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. In the embodiment of this application, the focal spot of the sound field can be determined first, and then the ultrasonic sound field conformer 2 can be set according to the focal spot. This application does not limit how to determine the focal spot of the sound field.

[0068] For example, the sound field can be computer-simulated by using acoustic detection instruments such as hydrophones and beam analyzers or according to the physical characteristics of the ultrasonic transducer, the size of the used focal spot can be measured, and the volume of the focal spot can be calculated and the central position of the focal spot can be determined.

[0069] Optionally, the shape of the ultrasonic sound field conformer 2 can be determined according to the shape of the focal spot of the sound field. For example, the shape of the ultrasonic sound field conformer 2 can be the same as or approximately the same as the shape of the focal spot.

[0070] As Figure 2 shown, in the embodiment of this application, the shape of the focal spot generated by the ultrasonic transducer 1 is ellipsoidal, so the shape of the ultrasonic sound field conformer 2 is also set to be ellipsoidal.

[0071] In other embodiments, the shape of the focal spot can be other shapes such as cylindrical, spherical, and three-dimensional polygon. Correspondingly, the shape of the ultrasonic sound field conformer 2 can also be set to be cylindrical, spherical, three-dimensional polygon, etc.

[0072] It should be understood that the shape of the ultrasonic sound field conformer 2 and the shape of the focal spot can also be different. For example, the shape of the focal spot is spherical, and at this time, the shape of the ultrasonic sound field conformer 2 can be set to be ellipsoidal or three-dimensional polygon, etc. This application does not limit this.

[0073] The size of the ultrasonic sound field conformer 2 can be determined according to the size of the focal spot. For example, the size of the ultrasonic sound field conformer 2 can be the same as, or close to (approximately the same as) the size of the focal spot.

[0074] Optionally, the size (or volume) of the ultrasonic sound field conformer 2 can be equal to, slightly larger than, or slightly smaller than the size (or volume) of the focal spot.

[0075] Optionally, the size of the ultrasonic sound field conformer 2 and the size of the focal spot can be proportional. For example, the size of the ultrasonic sound field conformer 2 can be 1.5 times, 2 times, 3 times, 5 times, etc. of the size of the focal spot, and this application does not limit this.

[0076] The focal length adapter 3 is used to fix the ultrasonic sound field conformer 2 at a preset position, and this preset position can be determined according to the position of the focal spot.

[0077] Optionally, this preset position can be determined according to the center position of the focal spot. For example, this preset position can be adjacent to the center position of the focal spot, or coincide with the center position of the focal spot.

[0078] That is to say, the center of the ultrasonic sound field conformer 2 can be set adjacent to the center position of the focal spot, or the center of the ultrasonic sound field conformer 2 can coincide with the center of the focal spot.

[0079] In the embodiment of this application, the shape of the ultrasonic sound field conformer 2 is the same as the shape of the focal spot, the size is the same, and the center position of the ultrasonic sound field conformer 2 coincides with the center position of the focal spot. Through the above settings, the ultrasonic energy can be maximally utilized, and thus the transfection efficiency can be improved.

[0080] In the embodiment of this application, the ultrasonic sound field conformer 2 is fixed at the preset position through the focal length adapter 3, and this application does not limit the specific form of the focal length adapter 3. In the embodiment of this application, the focal length adapter 3 is a collimator.

[0081] Optionally, in other embodiments, the focal length adapter 3 can also be other structures. For example, the focal length adapter 3 can be any one of a fixed bracket, a wire, a pull rope, etc.

[0082] As Figure 1 、 2 shown, in the embodiment of this application, 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. When the ultrasonic sound field conformer 2, the focal length adapter 3 and the ultrasonic transducer 1 are connected together, the ultrasonic sound field conformer 2 is located at this preset position.

[0083] That is to say, the focal length adapter 3 provided in the embodiments of the present application can play a role in focusing and positioning. When the ultrasonic sound field conformer 2, the focal length adapter 3 and the ultrasonic transducer 1 are connected together, the ultrasonic sound field conformer 2 can automatically be located at the preset position. Therefore, it is not necessary to manually adjust the position of the ultrasonic sound field conformer 2, thus simplifying the operation steps and ensuring the convenience and high efficiency of gene transfection.

[0084] In the embodiments of the present application, the ultrasonic sound field conformer 2 and the focal length adapter 3 are fixedly connected and form an integral structure through an integral molding process.

[0085] Through the above settings, there will be no relative displacement between the ultrasonic sound field conformer 2 and the focal length adapter 3. When the focal length adapter 3 is connected to the ultrasonic transducer 1, the ultrasonic sound field conformer 2 can automatically be located at the preset position. Since there is no need to perform a connection operation on 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 above two is avoided, thus further simplifying the operation steps and improving the positioning accuracy.

[0086] For example, the integral molding process can be injection molding.

[0087] For another example, the ultrasonic sound field conformer 2 and the focal length adapter 3 can also be integrally formed by 3D printing technology. At this time, the above two can be integrally formed by 3D printing technology with materials such as photosensitive resin, plastic, rubber, glass, metal, etc.

[0088] Optionally, in other embodiments, the ultrasonic sound field conformer 2 and the focal length adapter 3 can also be made of other materials such as glass, metal, plastic, etc., and the present application does not limit this.

[0089] Optionally, in other embodiments, the ultrasonic sound field conformer 2 and the focal length adapter 3 can be detachably connected.

[0090] As Figure 2 shown, in the embodiments of the present application, 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. 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.

[0091] The focal length adapter 3 is detachably connected to the ultrasonic transducer 1 through the connector 7, which can realize the flexible and simple connection of the two. At the same time, it is also convenient to replace and use different ultrasonic transducers to meet the needs of different experiments or treatments.

[0092] In the embodiments of the present application, the connector 7 can be the connection port between the ultrasonic transducer 1 and the focal length adapter 3. It is designed as a chute and is arranged on the inner wall surface at the lower end of the focal length adapter 3, and can be fixedly connected to the upper end of the ultrasonic transducer 1. In other embodiments, the connector 7 can also be other structures that can achieve the detachable connection between the ultrasonic transducer 1 and the focal length adapter 3, and the present application does not limit this.

[0093] As Figure 2 shown, the ultrasonic sound field conformer 2 is fixedly connected inside the focal length adapter 3. Specifically, the ultrasonic sound field conformer 2 is fixedly arranged on the inner wall surface of the top wall 32, and a sample loading port 4 is opened on the top wall 32. The sample loading port 4 is communicated with the opening of the ultrasonic sound field conformer 2. Thus, the complex of genes, cells and ultrasonic response particles can be put into the ultrasonic sound field conformer 2.

[0094] At this time, since the ultrasonic sound field conformer 2 is not sealed, during use, in order to avoid cell contamination, the sample loading port 4 can be sealed with a sealing film.

[0095] In other embodiments, the focal length adapter 3 can also be fixedly arranged on the peripheral wall 31, and correspondingly, the sample loading port 4 is opened on the peripheral wall 31.

[0096] In other embodiments, the sample loading port 4 may not be opened on the focal length adapter 3. After injecting the complex into the ultrasonic sound field conformer 2, the ultrasonic sound field conformer 2 can be fixedly arranged inside the focal length adapter 3.

[0097] Further, as Figure 1 、 2 shown,

[0098] 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 the height of the inner surfaces of other parts of the top wall 32, and the sample loading port 4 is opened in the concave structure 6.

[0099] By setting the concave structure 6, on the one hand, it is convenient for feeding and can prevent the complex from flowing into the external environment; on the other hand, the height of the inner surface of the concave structure 6 is lower than the height of the inner surfaces of other parts of the top wall 32. When there are bubbles, the bubbles will be displaced to other parts of the top wall 32. Thus, the propagation of ultrasonic waves will not be affected by the bubbles, avoiding the problem of energy attenuation, and further being beneficial to improving the efficiency of gene transfection. At this time, even if the sample loading port 4 is not provided, it can also play the role of removing bubbles.

[0100] Further, in order to conduct ultrasonic waves, a conduction medium can be filled in the focal length adapter 3, and the conduction medium can be any one of water, coupling liquid, PVA, etc.

[0101] Further, in order to ensure that the ultrasonic wave propagation is not affected by bubbles and avoid energy attenuation, an exhaust hole 5 can be opened on the top wall 32, and the air inside the focal length adapter 3 is discharged through the exhaust hole 5.

[0102] For example, before use, it should be checked whether there are bubbles inside the focal length adapter 3. If there are bubbles, the air should be discharged through the exhaust hole 5. The exhaust method is to inject a medium (most commonly water) using a 50 ml syringe until the bubbles disappear before ultrasonic irradiation can be carried out.

[0103] As Figure 1 shown, the gene transfection device provided by the embodiment of the present application further includes a holder 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.

[0104] Optionally, the holder 8 can be an iron stand lifting table, and components such as the ultrasonic transducer 1, the ultrasonic sound field conformer 2, and the focal length adapter 3 are fixed above it through the iron stand lifting table.

[0105] Optionally, a small hole can be opened at the center of the holder 8 to facilitate the signal transmission line 11 to pass through and connect with the ultrasonic transducer 1.

[0106] As Figure 1 shown, the gene transfection device provided by 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.

[0107] To meet different experimental and medical needs, in the embodiment of the present application, both the ultrasonic transducer 1 and the ultrasonic sound field conformer 2 include multiple ones. The multiple ultrasonic transducers 1 correspond to the multiple ultrasonic sound field conformers 2 one by one, and the sound fields of the ultrasonic waves emitted by each ultrasonic transducer 1 are different.

[0108] In this way, during use, it is possible to first determine which ultrasonic transducer 1 to select for work according to experimental and medical needs. After determining the ultrasonic transducer 1, it is possible to further select a matching ultrasonic sound field conformer 2 for work.

[0109] Optionally, when the ultrasonic sound field conformer 2 and the focal length adapter 3 are of an integrated structure, after determining the ultrasonic transducer 1, it is also possible to further select a matching focal length adapter 3 for work.

[0110] The research team where the inventor is located has used the gene transfection device provided by the embodiment of the present application for experimental verification and achieved efficient gene transfection of 293T cells using the sonoporation effect. The experimental steps are as follows:

[0111] 1. Prepare the gene transfection device in advance. Place the fixture 8 in the water inside the medium container 10, place the ultrasonic transducer 1 on it, and fit the corresponding focal length adapter 3. Remove the air bubbles through the vent hole 5. Start the ultrasonic signal outputter 9 and set its transfection parameters to a frequency of 1 - 3 MHz, a duty cycle of 10 - 50%, and an irradiation time of 0.1 - 10 minutes.

[0112] 2. Use a particle counting analyzer to calculate the number of prepared phospholipid vesicles.

[0113] 3. Take 5*10 5 -5*10 8 phospholipid vesicles + 0.5 - 5 μg plasmid and mix well to make a transfection complex, and let it stand at room temperature for 10 - 30 minutes.

[0114] 4. Digest the cells and count 10 - 20*10 4 cells per well. Add the prepared complex of phospholipid vesicles and plasmid. Put the complex into the ultrasonic field conformer 2 through the sample loading port 4 opened on the focal length adapter 3, seal the sample loading port 4 with a sealing film, and then perform irradiation according to the ultrasonic parameters designed in the experiment, paying attention to aseptic operation.

[0115] 5. After the operation, recover the cells from the ultrasonic field conformer 2, quickly add DMEM medium, spread them on a 24-well plate, incubate for 24 h, and observe the transfection situation with a fluorescence microscope after 24 h. The results of this experiment are as shown in Figure 4 (c).

[0116] To have a clearer understanding of the performance of the gene transfection device provided in the embodiments of the present application, the research team where the inventor is located also conducted a comparative experiment. Figure 4 It is a comparative diagram of observing the transfection situation with a fluorescence microscope under different experimental conditions.

[0117] Among them, Figure 4 (a) is the blank control group, with only cells, without the complex of phospholipid vesicles and genes, and the result is that no cells express the mCherry fluorescent protein. Figure 4 (b) is the group with the complex of phospholipid vesicles + genes, but ultrasonic irradiation is performed using the existing gene transfection device, and the result is that only a very small number of cells express the fluorescent protein; 4(c) is the group with the complex of phospholipid vesicles + genes, and ultrasonic irradiation is performed using the gene transfection device provided in the embodiments of the present application, and the result is that most cells express the fluorescent protein. The experimental results prove that the gene transfection device provided in the embodiments of the present application can improve the gene transport effect and has a high transfection efficiency.

[0118] As described above, it is only the specific implementation manner of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present application can easily think of changes or substitutions, which should all be covered within the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claimed rights.

Claims

1. An ultrasonic sound field conformal gene transfection device, characterized in that, Comprising: An ultrasonic transducer (1) for emitting ultrasonic waves; An ultrasonic sound field conformer (2) for accommodating a complex of genes, cells, and ultrasonic response particles, wherein the shape and size of the ultrasonic sound field conformer (2) are determined according to the sound field of the ultrasonic waves; A focal length adapter (3) for fixing the ultrasonic sound field conformer (2) at a preset position, the preset position being determined according to the position of the sound field of the ultrasonic waves; The shape of the ultrasonic sound field conformer (2) is the same as the shape of the focal spot of the sound field; The size of the ultrasonic sound field conformer (2) is the same as the size of the focal spot, or the size of the ultrasonic sound field conformer (2) is 1.5 times, 2 times, 3 times, or 5 times the size of the focal spot; The preset position is the central position of the focal spot; 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). When the ultrasonic sound field conformer (2), the focal length adapter (3), and the ultrasonic transducer (1) are connected together, the ultrasonic sound field conformer (2) is located at the preset position.

2. The gene transfection device according to claim 1, wherein The ultrasonic sound field conformer (2) and the focal length adapter (3) form an integral structure through an integral molding process.

3. The gene transfection device according to claim 2, wherein The ultrasonic sound field conformer (2) and the focal length adapter (3) are integrally formed by 3D printing technology.

4. The gene transfection device according to any one of claims 1-3, characterized in that, The ultrasonic sound field conformer (2) is fixedly connected inside the focal length adapter (3).

5. The gene transfection device according to claim 4, characterized in that The focal length adapter (3) includes a top wall (32). The ultrasonic sound field conformer (2) is fixedly arranged on the inner wall surface of the top wall (32). A sample loading port (4) is formed on the top wall (32), and the sample loading port (4) is communicated with the opening of the ultrasonic sound field conformer (2).

6. The gene transfection device according to claim 5, wherein An exhaust hole (5) is formed on the top wall (32).

7. The gene transfection device according to claim 5 or 6, characterized in that, 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 the height of the inner surfaces of other parts of the top wall (32). The sample loading port (4) is formed on the concave structure (6).

8. The gene transfection device according to claim 5, wherein The focal length adapter (3) further includes 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). The other end of the peripheral wall (31) is provided with a connector (7). The focal length adapter (3) is detachably connected to the ultrasonic transducer (1) through the connector (7).

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

10. The gene transfection device according to any one of claims 1-3, characterized in that, The ultrasonic response particles are ultrasonic microbubbles.

11. The gene transfection device according to any one of claims 1 to 3, characterized in that, Both the ultrasonic transducer (1) and the ultrasonic sound field conformer (2) include multiple ones. The multiple ultrasonic transducers (1) correspond to the multiple ultrasonic sound field conformers (2) one by one, and the sound fields of the ultrasonic waves emitted by each ultrasonic transducer (1) are different.

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