AU2010365282B2

AU2010365282B2 - Ultrasonic microbubble target-locating controlled releasing drug/gene device and target transfer method

Info

Publication number: AU2010365282B2
Application number: AU2010365282A
Authority: AU
Inventors: Pan Li; Zhiyu Ling; Haitao Ran; Zhigang Wang; Siyuan Yan; Qunxia Zhang; Yuanyi Zheng
Original assignee: Chongqing Ronghai Medical Ultrasound Ind Ltd; Chongqing Ronghai Engineering Research Center of Ultrasonic Medicine Co Ltd
Current assignee: Chongqing Ronghai Engineering Research Center of Ultrasonic Medicine Co Ltd
Priority date: 2010-12-08
Filing date: 2010-12-08
Publication date: 2014-02-27
Anticipated expiration: 2030-12-08
Also published as: AU2010365282A1; WO2012075602A1

Abstract

An ultrasonic microbubble target-locating controlled releasing drug/gene device and a method used for realizing target transfer of microbubble contrast agent combined with drug/gene by ultrasonic are disclosed. The ultrasonic microbubble target-locating controlled releasing drug/gene device comprises ultrasonic triggering/treating unit, monitoring unit, movement system connected with the ultrasonic triggering/treating unit and the monitoring unit, driving unit connected with the ultrasonic triggering/treating unit, computer control unit respectively connected with the movement system, the driving unit and the monitoring unit. The computer control unit comprises input system, computer center and output system.

Description

1 ULTRASONIC MICROBUBBLE TARGETED-LOCALIZATION DRUG/GENE CONTROLLED RELEASE DEVICE AND TARGETED TRANSFER METHOD FIELD OF THE INVENTION The present invention belongs to the field of biomedical engineering technology, and particularly relates to an ultrasonic microbubble targeted-localization drug/gene controlled release device and a method for realizing targeted transfer of a drug/gene using ultrasound by combining a microbubble contrast agent with the drug/gene. BACKGROUND OF THE INVENTION Drug/gene transfer is a key technique for making full use of the intrinsic biological effects of a drug/gene. Nowadays, there are viral drug/gene transfer vectors and non-viral drug/gene transfer vectors in general, such as direct injection of drug/gene, liposome mediated method, receptor mediated method, particle bombardment, electroporation, etc. In the gene transfer technology, the existing viral vectors have the advantages of high infection rate of gene, but have such problems as low safety and immune response of organism to viruses; while the existing non-viral vectors such as plasmids are safe, but low in infection rate. Like a gene drug, intravenous injection for transfer of protein drugs, polypeptide drugs and chemotherapeutic drugs also have the disadvantages of poor targeting ability, small amount of drug/gene to specific tissue, and low efficiency; while local injection of drug/gene has such problems as limited application range and invasion. At present, there are many problems to be solved in drug/gene transfer technology, especially lack of drug/gene transfer systems which are safe and effective and have tissue specificity and targeting ability. SUMMARY OF THE INVENTION Accordingly, the present invention provides an ultrasonic microbubble targeted localization drug/gene controlled release device comprising an ultrasonic triggering/treating unit which realises targeted drug/gene transfer by using ultrasound to trigger destruction of microbubbles and a driving unit which drives the ultrasonic 2 triggering/treating unit to emit ultrasonic energy, wherein the ultrasonic microbubble targeted-localization drug/gene controlled release device further comprises a monitoring unit, a movement system and a computer control unit, the ultrasonic triggering/treating unit and the monitoring unit are connected to the movement system, the driving unit is connected to the ultrasonic triggering/treating unit, and the computer control unit is connected to the movement system, the driving unit and the monitoring unit respectively, wherein an opening is formed in the middle of the ultrasonic triggering/treating unit, and a monitoring probe of the monitoring unit goes through the opening and is fixed together with an ultrasonic triggering/treating applicator of the ultrasonic triggering/treating unit, ultrasound images acquired by the monitoring unit are used to monitor the targeted transfer and located controlled release of drug/gene-carried microbubble contrast agent, and the ultrasound images acquired by the monitoring unit are used to monitor the arrival and development of the microbubble contrast agent. Preferably, the computer control unit includes an input system, a computer center and an output system, the input system includes a keyboard, and input devices for information of the monitoring unit, information of the movement system and information of the driving unit, the computer center is composed of hardware and software, the hardware includes at least one type of information acquisition card/circuit interface, a CPU, at least one type of output circuit interface and a display device for displaying information such as sound and images, and the software includes a system software, a data/image processing software, and an application software easy to operate/display. Preferably, the software further comprises an application program connected to the CPU and the or each type of output circuit interface. This is a new drug/gene transfer method which is non-invasive and has the advantages of high efficiency and safety, simple operation and good targeting ability and repeatability. In such drug/gene transfer method, a specific drug/gene is combined with a microbubble contrast agent which is a new drug/gene transfer vector, the obtained drug/gene-carried microbubble contrast agent is subjected to peripheral vascular 2a injection, and then the gene-carried microbubbles are destroyed with ultrasound, so that the microbubbles can release the drug/gene in located specific tissue. Another advantage 3 is that, this is a dedicated device for targeted-localization drug/gene controlled release, especially an integrated device having locating, monitoring, diagnosing and treating functions. Aiming at the above deficiencies existing in the prior art, the advantage of the present invention is to provide an ultrasonic microbubble targeted-localization drug/gene controlled release device capable of increasing drug/gene transfer efficiency and locating and monitoring drug/gene transfer. Another advantage of the present invention is to provide a method for realizing targeted transfer of a drug/gene using ultrasound by combining a microbubble contrast agent with the drug/gene. Preferably, the system software and the data/image processing software constitute an application program. Parameters are set by operating the software, information is transmitted to the movement system and the driving unit via the output circuit interface(s), the driving unit drives the ultrasonic triggering/treating unit to emit ultrasonic energy, the monitoring unit is used to monitor treatment and feed back information to the information acquisition card(s), the information is transmitted to the application program via the data/image processing software, the application program automatically processes the information in corresponding ways or reminds an operator to operate correspondingly, tissue characteristics are recognized and showed in the forms of numerals, colors, characters, etc. Wherein, a B-mode ultrasonic scanner is preferably used as the monitoring unit. Preferably, the software further comprises an application program connected to the CPU and the output circuit interface(s). The application program includes a data/image processing software and an application software, and further includes a data/image processing software and an application software which are connected to the display device for evaluating effect of targeted drug/gene transfer. In operation, the application program controls the ultrasonic triggering/treating unit to act on a tissue by calculating power and acting time, timely image information (in the forms of numerals, colors, characters, etc.) before and after the action is showed via the display device, changes of 4 the image information are used to judge whether the effect is satisfying, and the power and the acting time are adjusted properly through the application software if the effect is not satisfying, so as to continue to act on the target tissue until achieving a satisfying effect. The device may comprise a cooling unit connected to the ultrasonic triggering/treating unit, so as to dissipate the heat generated by the ultrasonic triggering/treating unit in time to prolong the service life thereof. Preferably, the monitoring unit and the ultrasonic triggering/treating unit are fixed together by several optional means. Preferably, an opening is formed in the middle of the ultrasonic triggering/treating unit, and a monitoring probe of the monitoring unit goes through the opening and is fixed together with an ultrasonic triggering/treating applicator of the ultrasonic triggering/treating unit. The fixing of the monitoring unit and the ultrasonic triggering/treating unit guarantees that an acting region of the ultrasonic triggering/treating unit can be indicated accurately in a monitor image. Therefore, when the device is in use, changes of the images of the acting region can be observed accurately and effect evaluation can be performed timely. The device can realize targeted drug/gene transfer by using ultrasound to trigger destruction of microbubbles, and perform effect evaluation of targeted drug/gene transfer, so as to achieve integration of diagnosis, treatment and evaluation. It is proved by using New Zealand white rabbits as experimental subjects that the device of the present invention can markedly increase drug/gene transfer efficiency. A method for realizing targeted transfer of a drug/gene using ultrasound by combining a microbubble contrast agent with the drug/gene comprises the following steps: (1) loading the microbubble contrast agent with the drug/gene to form a drug/gene-carried microbubble contrast agent; (2) monitoring the targeted transfer and located controlled release of the drug/gene carried microbubble contrast agent through ultrasound images acquired by a monitoring unit of the aforesaid ultrasonic microbubble targeted-localization drug/gene controlled release device, that is, subjecting the drug/gene-carried microbubble contrast agent to intravenous injection; (3) monitoring the microbubble contrast agent with the monitoring unit; (4) driving an ultrasonic triggering/treating unit to emit ultrasound to destroy microbubbles after the microbubble contrast agent arrives at a target tissue and is developed; (5) acting on the target tissue directly with the ultrasound after the microbubbles are destroyed; (6) controlling power and time of ultrasound emission with a computer control unit until the targeted transfer and located controlled release of the drug/gene are completed. Preferably, after the step (5), the following steps for evaluating the effect after the drug/gene transfer are performed: (5-1) acquiring real-time image information (in the forms of numerals, colors, characters, etc.) of the target tissue, which is obtained by a monitoring unit, by a computer control unit via an image acquisition card, after the microbubbles are destroyed by the ultrasound; (5-2) transmitting the above real-time image information of the target tissue to a display device; (5-3) observing the arrival of the drug/gene-carried microbubble contrast agent at the target tissue, and the transfection and expression thereof via the display device; (5-4) simultaneously, transmitting ultrasound image information in the forms of numerals, colors, characters, etc., which is obtained by the monitoring unit, to an application system via an application software for comparison and evaluation of acting effect; (5-5) adjusting parameters related to the ultrasound set in the application software according to the result of the evaluation of acting effect, that is, judging whether the effect is satisfying according to changes of the image 5 information, adjusting the application software to set the parameters of the dose and the acting time of the ultrasound if the effect is not satisfying, and continuing to act on the target tissue until achieving the targeted transfer and located controlled release. In order to accurately control the dose and the acting time of the ultrasound, corresponding parameters are set through the application software before the driving unit drives the ultrasonic triggering/treating unit to emit the ultrasound for destroying the microbubbles; and then the ultrasound is emitted. Wherein, frequency range of the emitted ultrasound set in the ultrasonic triggering/treating unit is 20 KHz-2 MHz, preferably 1 MHz; ultrasonic intensity range is 0.25 W/cm 2 -3 W/cm 2 , preferably 0.5 W/cm 2 ; and the acting time is 0.25 min-3 min, preferably 1 min. Preferably, the drug/gene-carried microbubble contrast agent formed by combining the microbubble contrast agent with the drug/gene in the step (1) to is obtained by attaching the drug/gene to the surfaces of the microbubble contrast agent, that it, mixing a selected drug/gene and a lipid microbubble contrast agent in the volume ratio of (3-10) to 10 and attaching the drug/gene to the surfaces of the microbubbles under the function of electrostatic adsorption. The aforesaid drug/gene-carried microbubble contrast agent is obtained by attaching purchased contrast agent or homemade contrast agent and the gene to the surfaces of the microbubble contrast agent. One method for producing the drug/gene-carried microbubble contrast agent comprises mixing the drug/gene with the purchased contrast agent in the volume ratio of (3-10) to 10, and attaching the drug/gene to the surfaces of the microbubbles under the function of electrostatic adsorption. Preferably, the other method for producing the drug/gene-carried microbubble contrast agent of step (1) from the homemade contrast agent comprises wrapping the drug/gene in the microbubbles, that is, mixing fluorocarbon gas, which is in liquid state in low temperature, with the drug/gene in the volume ratio of (3-5) to 1, subjecting them to ultrasonic 6 7 vibration to form microspheres composed of lipid material with the fluorocarbon gas and the drug/gene wrapped therein, increasing the temperature to 37*C-45*C to change the microspheres into microbubbles, and washing away the drug/gene which is not wrapped in the lipid material with a buffer solution (such as PBS buffer solution) to obtain the drug/gene-carried microbubble contrast agent. Preferably, in the step (1), the microbubble contrast agent is attached to the drug/gene to form the drug/gene-carried microbubble contrast agent, antibodies for specific tissue antigens or ligands for specific receptors are connected to the surfaces of the microbubbles, and various antibodies are mixed in liquid during ultrasonic vibration to form the ultrasonic microbubble contrast agent having targeting function. Preferably, the aforesaid drug refers to a chemotherapeutic drug for tumors, an antibiotic, or various protein drugs and polypeptide drugs; and the aforesaid gene refers to a vascular endothelial growth factor (VEGF), a hepatocyte growth factor (HGF), a fibroblast growth factor (FGF), etc., and marker genes such as a green fluorescent protein gene (GFP) and a P-galactosidase gene. The method is non-invasive and simple, and can be applied repeatedly. BRIEF DESCRIPTION OF THE DRAWINGS Fig. I is a block diagram showing the structure principle of an ultrasonic microbubble targeted-localization drug/gene controlled release device of the present invention; Fig. 2 is a three-dimensional diagram showing the structure relation of an ultrasonic monitoring unit 3 and an ultrasonic triggering/treating unit 2 of the present invention (when a housing is disposed horizontally); Fig. 3 is a three-dimensional diagram showing the structure relation of the ultrasonic monitoring unit 3 and the ultrasonic triggering/treating unit 2 of the present invention (when the housing is disposed vertically); Fig. 4 is a block diagram showing the structure principle of a computer control unit 4 of the present invention; Fig. 5A is a digital subtract angiogram of a control group of an experiment on promotion of angiogenesis by ultrasonic gene-carried microbubbles of the present invention; Fig. 5B is a digital subtract angiogram of an experimental group of the experiment on promotion of angiogenesis by ultrasonic gene-carried microbubbles of the present invention. Wherein: 1-target tissue, 2-ultrasonic triggering/treating unit, 20-ultrasonic triggering/treating applicator, 3-monitoring unit, 30-monitoring probe, 4-driving unit, 5-movement system, 6-computer control unit, 61-information acquisition card/circuit interface, 62-CPU, 63-application program, 64-system software, 65-data/image processing software, 66-application software, 67-display device, 68-output circuit interface, 7-cooling unit, 8-connecting tube, 9-housing, 10, 11, 12, 13-opening, 14-sound transmission film Detailed Description of the Preferred Embodiments The present invention will be further described in detail hereinafter in connection with the embodiments and the drawings. The following embodiment is a non-restrictive embodiment of the present invention. As show in Fig. 1, the ultrasonic microbubble targeted-localization drug/gene controlled release device of the present invention comprises an ultrasonic triggering/treating unit 2, a monitoring unit 3, a driving unit 4 used to drive the ultrasonic triggering/treating unit 2 to emit ultrasound, a movement system 5, a computer control unit 6 and a cooling unit 7, wherein, the computer control unit 6 is connected to the monitoring unit 3, the driving unit 4 and the movement system 5 respectively, and the driving unit 4 and the cooling unit 7 8 are connected to the ultrasonic triggering/treating unit 2 respectively. The ultrasonic triggering/treating unit 2 is used to trigger destruction of microbubbles so as to realize targeted drug/gene transfer and treatment of a target tissue 1, and the monitoring unit 3 is used to monitor the treating of the target tissue 1. The ultrasonic triggering/treating unit 2 includes an ultrasonic triggering/treating applicator 20, and the monitoring unit 3 includes a monitoring probe 30. In this embodiment, a B-mode ultrasonic scanner is used as the monitoring unit 3. As shown in Fig. 4, the computer control unit 6 includes an input system, a computer center and an output system, an information acquisition card/circuit interface 61 in the input system is connected to a CPU 62 of the computer center, the CPU 62 is connected to an application program 63 which is connected to a display device 67 of the output system, the information acquisition card/circuit interface 61 is connected to the monitoring unit 3, and the application program 63 further comprises a data/image processing software 65 and an application software 66 and is connected to the movement system 5 and the driving unit 4 via an output circuit interface 68. Certain technical parameters can be set in the application software 66 to make the ultrasonic triggering/treating applicator 20 act on the target tissue 1 with certain power for certain time, image information (in the forms of numerals, colors, characters, etc.) before and after the action, which is acquired by the information acquisition card/circuit interface 61, is showed in the display device, changes of the image information are used to judge whether a satisfying effect is achieved, and the power and the acting time are adjusted through the application software 63 if the satisfying effect is not achieved, so as to continue to act on the target tissue 1 until achieving the satisfying effect. Wherein, the ultrasound emitted by the ultrasonic triggering/treating unit 2 is focused/non-focused therapeutic/diagnostic ultrasound and is pulse waves or continuous waves, its frequency may be 20 KHz-2 MHz and its 9 intensity may be 0.25 W/cm 2 -3 W/cm 2 . The monitoring probe 30 (that is, a B-mode ultrasonic probe) of the monitoring unit 3 is integrated with the ultrasonic triggering/treating applicator 20 of the ultrasonic triggering/treating unit 2. As shown in Figs. 2-3, an opening is formed in the middle of the ultrasonic triggering/treating applicator 20, and the monitoring probe goes through the opening and is fixed in the opening. As shown in Figs. 2-3, a housing 9 is arranged outside the ultrasonic triggering/treating unit 2 to surround it, and is fixed to the ultrasonic triggering/treating applicator 20 by a bolt via an opening 11, a connecting tube 8 is arranged under the monitoring probe 30, and is fixed to the housing 9 by a bolt via an opening 12. With such configuration, the ultrasonic triggering/treating applicator 20 and the monitoring probe 30 are fixed together via the housing 9. Openings 13 for cables which connect the ultrasonic triggering/treating applicator 20 to the driving unit 4 and the computer control unit 6 are provided in the housing 9, and also serve as cooling passages that communicate with the cooling unit 7. A cable which connects the monitoring unit 3 with the computer control unit 6 is mounted in the connecting tube 8, and a motor for driving the movement system 5 to move is mounted in the connecting tube 8 as well. As shown in Fig. 3, the ultrasonic triggering/treating applicator 20 includes an ultrasonic transducer, an opening 10 is formed in the ultrasonic transducer and serves as a water inlet and drainage passage, and a sound transmission film 14 is fixed at an end of the ultrasonic triggering/treating applicator 20 by means of bonding or by other means. When the device of the present invention is in use, the monitoring probe 30 and the ultrasonic triggering/treating applicator 20 move along the direction towards the target tissue 1, and the ultrasonic triggering/treating applicator 20 emits ultrasound to trigger destruction of microbubbles to realize targeted drug/gene transfer so as to treat the target tissue 1. 10 When the device of the present invention is used to destroy the microbubbles with ultrasound for realizing the targeted drug/gene transfer, microbubble contrast agent is combined with a drug/gene at first to form drug/gene-carried microbubble contrast agent, the drug/gene-carried microbubble contrast agent is then subjected to peripheral vascular injection, and finally targeted transfer of the drug/gene-carried microbubble contrast agent is monitored via ultrasound images obtained by the monitoring unit 3 and ultrasound is emitted to destroy the drug/gene-carried microbubbles so as to make the microbubbles to release in located specific tissue. The method of using the device of the present invention to destroy the microbubbles with ultrasound for realizing the targeted drug/gene transfer comprises: (1) attaching the drug/gene to the surfaces of the microbubble contrast agent: mixing a selected drug/gene and a lipid microbubble contrast agent in the volume ratio of (3-10) to 10 (to make the amount of the drug/gene in the mixture to be 0.1 mg/ml-1 mg/ml), and attaching the drug/gene to the surfaces of the microbubbles under the function of electrostatic adsorption so as to form the microbubble contrast agent whose surfaces are attached with the drug/gene; Or, wrapping the drug/gene in the microbubbles: mixing fluorocarbon gas, which is in liquid state in low temperature, with the drug/gene in the volume ratio of (3-5) to 1, subjecting them to ultrasonic vibration to form microspheres composed of lipid material with the fluorocarbon gas and the drug/gene wrapped therein, increasing the temperature to 37C-45*C to change the microspheres into microbubbles, and washing away the drug/gene which is not wrapped in the lipid material with a PBS buffer solution so as to obtain the drug/gene-carried microbubble contrast agent. (2) monitoring the targeted transfer and located controlled release of the drug/gene-carried microbubble contrast agent via the ultrasound images acquired by the monitoring unit 3: setting ultrasonic intensity to be 0.5 W/cm 2 frequency to be 1 MHz and acting time to be 1 min in the application software 11 66, subjecting the drug/gene-carried microbubble contrast agent to intravenous injection, monitoring the microbubble contrast agent via the ultrasound images acquired by the monitoring unit 3 (B-mode ultrasonic scanner), and driving the ultrasonic triggering/treating applicator 20 of the ultrasonic triggering/treating unit 2 by the driving unit 4 to emit ultrasound to destroy the microbubbles after the microbubble contrast agent arrives at the target tissue 1 and is developed. Cavitation effect and sonoporation happened after the microbubbles are destroyed can lead to rupture of capillaries and generation of pores on cell membranes, and greatly increase transfection efficiency of the gene in the target tissue 1; (3) evaluating the effect after the drug/gene is transferred: after the microbubbles are destroyed with ultrasound, comparing through the application software the image information which is acquired by the information acquisition card/circuit interface 61 and displayed in the display device and shows the arrival of the drug/gene-carried microbubble contrast agent at the target tissue and the transfection and expression thereof, adjusting the application software 66 to set related parameters according to the changes of the image information, and continuing to act on the target tissue 1 until achieving the targeted transfer and located controlled release. During the whole working process of the device of the present invention, the cooling unit 7 is used to provide cooling water to cool the heat generated by the ultrasonic triggering/treating unit 2. Figs. 5A-5B are comparison diagrams of an experiment which proves that ultrasonic gene-carried microbubbles can markedly promote angiogenesis. The experiment is carried out as follows: adopting healthy New Zealand white rabbits weighing 2.5 kg-3 kg as experimental subjects, subjecting the rabbits to muscle anaesthesia with SUMIANXIN (0.1 ml/kg-0.15 ml/kg), removing the hair of the rabbits with 8% Na 2 S, laying the rabbits on their backs on an operating board, fixing them on the operating board, and performing femoral artery ligation and femoral artery branch ligation of the left 12 lower limbs of the rabbits to form models of occlusive vascular disease of lower limbs. The rabbits are divided randomly into an experimental group and control groups after the models are formed, the experimental group is an ultrasonic microbubble treatment group (A), and the control groups include a blank control group (B), a mere ultrasonic irradiation group (C) and a mere microbubble treatment group (D). The microbubbles used in the experiment are an albumin microbubble contrast agent with concentration of 8.3x108 microbubbles/mi and size of 2.7±0.8 pm. After the models are formed, Group A are injected locally with the mixture of albumin microbubbles and pcDNA3.1NEGF165 plasmid in the amount of 25 pg, and irradiated with ultrasound, with the emission frequency of 1 MHz, the ultrasonic intensity of 2.0 W/cm 2 and the irradiation duration of 1 min as the parameters of the ultrasound. Group B are not treated with ultrasound and microbubbles, and serve as the blank control group. Without being treated with microbubbles, Group C are only irradiated with ultrasound whose parameters are the same as those of Group A. After the models are formed, Group D, namely the mere microbubble treatment group, are injected locally with 25 pg of pcDNA3.1NEGF165 plasmids through skeletal muscles, without being irradiated with ultrasound. Four weeks later, cannulas are inserted into the abdominal aortas of the rabbits, 2 ml of 76% meglucamine diatrizoate contrast agent is injected into the rabbits within 1 s under increased pressure, and continuous cinematography is used to record angiographic results in picture rate of 25 frame/s for observing formation of new vessels and collateral circulation. The results show that there are only a few new vessels in the skeletal muscles of the control groups (as shown in Fig. 5A), while the number of the new vessels in the skeletal muscles of the experimental group, namely the ultrasonic microbubble treatment group, increases markedly relative to that of the control groups (as shown in Fig. 5B), which indicates that destruction of VEGF165 gene-carried microbubbles by ultrasound can greatly promote angiogenesis in ischemic skeletal muscles. 13 14 The microbubble contrast agent may be homemade contrast agent, purchased microbubble contrast agent such as Sonovue made in Italy, Optison and Albunex made in U.S.A. and Levovist made in Germany, or homemade ultrasound contrast agent. The purchased or homemade microbubble contrast agent is attached with a drug/gene, and antibodies for specific tissue antigens or ligands for specific receptors are connected optionally to the surfaces of the microbubbles so as to greatly enhance the targeting function of the microbubbles. Various antibodies, such as anti-CD34, anti-ICAM, anti-E selectin and anti-P-selectin, are mixed in liquid during ultrasonic vibration to form the ultrasonic microbubble contrast agent having targeting function. The gene involved in the present invention refers to an available existing gene, which includes various growth factors such as a vascular endothelial growth factor (VEGF), a hepatocyte growth factor (HGF) and a fibroblast growth factor (FGF), and marker genes such as a green fluorescent protein gene (GFP) and a D-galactosidase gene. The drug suitable for this embodiment includes all bioactive substances such as chemotherapeutic drugs for tumors, antibiotics, various protein drugs and polypeptide drugs. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference to any prior art in this specification is not and should not be taken as an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge in Australia.

Claims

1. An ultrasonic microbubble targeted-localization drug/gene controlled release device comprising an ultrasonic triggering/treating unit and a driving unit, wherein the ultrasonic microbubble targeted-localization drug/gene controlled release device further comprises a monitoring unit, a movement system and a computer control unit, the ultrasonic triggering/treating unit and the monitoring unit are connected to the movement system, the driving unit is connected to the ultrasonic triggering/treating unit, and the computer control unit is connected to the movement system, the driving unit and the monitoring unit respectively, wherein an opening is formed in the middle of the ultrasonic triggering/treating unit, and a monitoring probe of the monitoring unit goes through the opening and is fixed together with an ultrasonic triggering/treating applicator of the ultrasonic triggering/treating unit, ultrasound images acquired by the monitoring unit are used to monitor the targeted transfer and located controlled release of drug/gene-carried microbubble contrast agent, and the ultrasound images acquired by the monitoring unit are used to monitor the arrival and development of the microbubble contrast agent.

2. The ultrasonic microbubble targeted-localization drug/gene controlled release device according to claim 1, wherein the computer control unit includes an input system, a computer center and an output system, the input system includes a keyboard, and input devices for information of the monitoring unit, information of the movement system and information of the driving unit, the computer center is composed of hardware and software, the hardware includes at least one type of information acquisition card/circuit interface, a CPU, at least one type of output circuit interface and a display device for displaying information such as sound and images, and the software includes a system software, a data/image processing software, and an application software easy to operate/display.

3. The ultrasonic microbubble targeted-localization drug/gene controlled release device according to claim 2, wherein the software further comprises an application program connected to the CPU and the or each type of output circuit interface. 16

4. The ultrasonic microbubble targeted-localization drug/gene controlled release device according to claim 3, wherein the application program includes a data/image processing software and an application software, which are connected to the display device for evaluating effect of targeted drug/gene transfer.

5. The ultrasonic microbubble targeted-localization drug/gene controlled release device according to any one of claim 1 to claim 4, further comprising a cooling unit connected to the ultrasonic triggering/treating unit.

6. The ultrasonic microbubble targeted-localization drug/gene controlled release device according to claim 5, wherein a B-mode ultrasonic scanner is used as the monitoring unit.

7. A method for realizing targeted transfer of a drug/gene using ultrasound by combining a microbubble contrast agent with the drug/gene through the ultrasonic microbubble targeted-localization drug/gene controlled release device according to any one of claim I to claim 6, comprising the following steps: (1) loading the microbubble contrast agent with the drug/gene to form a drug/gene-carried microbubble contrast agent; (2) monitoring the targeted transfer and located controlled release of the drug/gene-carried microbubble contrast agent through ultrasound images acquired by the monitoring unit of the ultrasonic microbubble targeted-localization drug/gene controlled release device and subjecting the drug/gene-carried microbubble contrast agent to intravenous injection; (3) monitoring the microbubble contrast agent with the monitoring unit; (4) driving the ultrasonic triggering/treating unit to emit ultrasound to destroy microbubbles after the microbubble contrast agent arrives at a target tissue and is developed; (5) acting on the target tissue directly with the ultrasound after the microbubbles are destroyed; and (6) controlling power and time of ultrasound emission with the computer control 17 unit until the targeted transfer and located controlled release of the drug/gene are completed.

8. The method for realizing targeted transfer of a drug/gene using ultrasound by combining a microbubble contrast agent with the drug/gene according to claim 7, wherein after the step (5), the following steps for evaluating the effect after the drug/gene transfer are performed: (5-1) acquiring real-time image information of the target tissue, which is obtained by the monitoring unit, by the computer control unit via an image acquisition card, after the microbubbles are destroyed by the ultrasound; (5-2) transmitting the above real-time image information of the target tissue to a display device; (5-3) observing the arrival of the drug/gene-carried microbubble contrast agent at the target tissue, and the transfection and expression thereof via the display device; (5-4) simultaneously, transmitting the ultrasound image information in the forms of numerals, colors and characters obtained by the monitoring unit, to the application system via the application software for comparison and evaluation of acting effect; (5-5) adjusting the parameters related to the ultrasound set in the application software according to the result of the evaluation of acting effect, and continuing to act on the target tissue until achieving the targeted transfer and located controlled release.

9. The method for realizing targeted transfer of drug/gene using ultrasound by combining a microbubble contrast agent with the drug/gene according to claim 8, wherein frequency range of the ultrasound emitted by the ultrasonic triggering/treating unit is 20 KHz-2 MHz, ultrasonic intensity range is 0.25 W/cm 2 -3 W/cm 2 , and acting time is 0.25 min-3 min.

10. The method for realizing targeted transfer of drug/gene using ultrasound by combining a microbubble contrast agent with the drug/gene according to any one of claim 7 to claim 9, wherein the drug/gene-carried microbubble contrast agent formed by combining the microbubble contrast agent with the drug/gene in the step (1) is obtained by attaching the drug/gene to surfaces of the microbubble contrast agent and mixing a selected drug/gene 18 and a lipid microbubble contrast agent in the volume ratio of (3-10) to 10 and attaching the drug/gene to the surfaces of the microbubbles under the function of electrostatic adsorption.

11. The method for realizing targeted transfer of a drug/gene using ultrasound by combining a microbubble contrast agent with the drug/gene according to any one of claim 7 to claim 10, wherein the drug/gene-carried microbubble contrast agent formed by combining the microbubble contrast agent with the drug/gene in the step (1) is obtained by wrapping the drug/gene in the microbubbles and mixing fluorocarbon gas, which is in liquid state in low temperature, with the drug/gene in the volume ratio of (3-5) to 1, subjecting them to ultrasonic vibration to form microspheres composed of lipid material with the fluorocarbon gas and the drug/gene wrapped therein, increasing the temperature to 37 0 C-45 0 C to change the microspheres into microbubbles, and washing away the drug/gene which is not wrapped in the lipid material with a buffer solution to obtain the drug/gene-carried microbubble contrast agent.

12. The method for realizing targeted transfer of a drug/gene using ultrasound by combining a microbubble contrast agent with the drug/gene according to any one of claim 7 to claim 11, wherein in the step (1), the microbubble contrast agent is attached to the drug/gene to form the drug/gene-carried microbubble contrast agent, antibodies for specific tissue antigens or ligands for specific receptors are connected to the surfaces of the microbubbles, and various antibodies are mixed in liquid during ultrasonic vibration to form the ultrasonic microbubble contrast agent having targeting function.

13. The method for realizing targeted transfer of a drug/gene using ultrasound by combining a microbubble contrast agent with the drug/gene according to any one of claim 7 to claim 12, wherein the drug includes any one or more of the following: a chemotherapeutic drug for tumors; an antibiotic; various protein drugs and polypeptide drugs; and the gene includes any one or more of the following: vascular endothelial growth factor (VEGF); a hepatocyte 19 growth factor (HGF); a fibroblast growth factor (FGF); a green fluorescent protein gene (GFP); and a -galactosidase gene.

14. An ultrasonic microbubble targeted-localization drug/gene controlled release device substantially as hereinbefore described with reference to any one of the accompanying drawings.