Acoustic-magnetic dual-mode diagnosis and treatment agent as well as preparation method and application thereof

Abstract

The invention provides an acoustic-magnetic dual-mode diagnosis and treatment agent as well as a preparation method and application thereof, and belongs to the technical field of preparation of tumor diagnosis and treatment agents. The acoustic-magnetic dual-mode diagnosis and treatment agent is surface-modified magnetic nanoparticles which contain nano balloon genes and are subjected to Escherichia coli induced expression; the nucleotide sequence of the nano-balloon gene is a sequence shown as NCBI: KC601845.1, escherichia coli in the acoustic-magnetic dual-mode diagnosis and treatment agent as a diagnosis and treatment agent carrier has an active targeting property on a tumor hypoxic region, and bacteria contain a nano-balloon and have an acoustic response characteristic; the escherichia coli surface modified magnetic nanoparticles have a magnetic response characteristic. In acoustic-magnetic dual-mode imaging, firstly, on a real-time ultrasonic contrast monitoring image, a nano air bag expressed in bacteria can generate an enhanced tumor ultrasonic echo signal; secondly, under the action of an external changing magnetic field, the magnetic nanoparticles on the surfaces of the bacteria can generate an enhanced tumor magnetic vibration imaging effect, so that more efficient tumor diagnosis and / or treatment can be realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of tumor diagnosis and treatment agent preparation, and particularly relates to a sound-magnetic dual-mode diagnosis and treatment agent and a preparation method and application thereof. BACKGROUND

[0002] Medical imaging technology is one of the indispensable tools for modern medical diagnosis. As an important auxiliary material for image diagnosis, contrast agents play an important role in improving image quality, clarifying lesion location and degree, etc. Existing contrast agents can be a compound or a micro-nano structure. Their main role is to increase or decrease the signal characteristics of tissues or organs in medical images, thereby improving the contrast and clarity of the images.

[0003] In X-ray and CT images, contrast agents usually contain high-density or high-atomic-number substances such as iodine, barium, bismuth, etc. These substances can absorb more X-rays, making the surrounding tissues or organs appear bright in the image. In MRI images, contrast agents are usually a complex containing metal ions (such as manganese, iron, gadolinium, etc.). These metal ions have special magnetic properties and can change the magnetic behavior of surrounding water molecules, thereby affecting the intensity and contrast of the MRI signal. In ultrasound images, contrast agents are mainly composed of encapsulated microbubbles (microbubbles for short) with a diameter of 2-5 microns. Microbubbles are composed of an inert gas (such as sulfur hexafluoride) core and a phospholipid or albumin outer membrane. Compared with human tissues, the gas core of microbubbles has stronger echo characteristics and can achieve enhanced visualization in ultrasound images.

[0004] In recent years, with the rapid development of biomedical imaging technology, targeted contrast agents have attracted widespread attention due to their ability to improve the specificity and accuracy of image diagnosis. Targeted contrast agents are combined with specific biological ligands (such as antibodies, peptides or aptamers) on the surface of the contrast agent. During the in vivo circulation and distribution of the contrast agent, these biological ligands can recognize and bind to specific molecules or structures on the target tissue or cells; thus, the signal of the contrast agent increases or decreases on the target tissue or cells, thereby achieving targeted recognition of the target tissue or cells.

[0005] However, the existing medical imaging contrast agent still has technical defects in targeting specific tissues or cells, mainly in the following three aspects: (1) non-specific targeting is still significant, the existing targeted medical imaging contrast agent still has significant non-specific distribution in special tissues after being injected into the body (for example, nanoparticles tend to accumulate and be eliminated in the liver region), which limits the accuracy of targeted diagnosis and treatment; (2) the specific targeting is still inefficient, the targeting efficiency of the existing targeted medical imaging contrast agent on the target tissue or cell is still not high enough, the affinity between the contrast agent and the specific target is not enough, or the contrast agent cannot effectively reach the target area due to the complex biological environment in the body; (3) the stability in the body is still not enough, the combination of some contrast agents and their specific biological ligands may lose stability in the body due to various factors such as enzyme degradation, oxidation, high-speed blood flow impact, etc. Such instability may cause the contrast agent to separate from its specific biological ligand, thereby losing the targeting ability. Therefore, it is a technical problem to be solved at present to provide a targeted acoustic-magnetic dual-mode diagnosis and treatment agent for diagnosing and / or treating tumors, improving the efficiency of specific targeting of tumors and the treatment effect. SUMMARY

[0006] In view of this, the purpose of the present application is to provide an acoustic-magnetic dual-mode diagnosis and treatment agent and a preparation method and application thereof.

[0007] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions: The present application provides an acoustic-magnetic dual-mode diagnosis and treatment agent, which is a surface modified magnetic nanoparticle of E. coli induced expression containing a nanobubble gene; the nucleotide sequence of the nanobubble gene is the sequence shown in NCBI: KC601845.1.

[0008] Preferably, the E. coli is E. coli BL21.

[0009] The construction method of the E. coli containing the nanobubble gene comprises the following steps: The expression vector containing the nanobubble gene is transformed into E. coli to obtain the E. coli containing the nanobubble gene.

[0010] Preferably, the backbone vector of the expression vector is pBAD33.

[0011] Preferably, the magnetic nanoparticle includes a carboxyl magnetic nanoparticle or a streptavidin modified magnetic nanoparticle.

[0012] Preferably, the surface modified magnetic nanoparticle of the E. coli containing the nanobubble gene after induced expression includes any one of the following: (1) carboxyl magnetic nanoparticles are modified on the surface of the E. coli containing the nanobubble gene after induced expression by amide coupling method; (2) after the E. coli containing nanobubble gene induced expression, the surface modified biotinylated Sulfo-NHS is connected with the streptavidin modified magnetic nanoparticles; (3) after the E. coli containing nanobubble gene induced expression, the surface modified biotinylated antibody is connected with the streptavidin modified magnetic nanoparticles.

[0013] The application provides a preparation method of a sound-magnetic dual-mode enhanced contrast agent, which comprises any one of the following methods a-c: a. mixing carboxyl magnetic nanoparticles, EDC and Sulfo-NHS to obtain activated magnetic nanoparticles; then incubating the E. coli containing nanobubble gene after induced expression with the activated magnetic nanoparticles to obtain the sound-magnetic dual-mode enhanced contrast agent; b. incubating the E. coli containing nanobubble gene after induced expression with biotinylated Sulfo-NHS to obtain surface biotinylated bacteria a, then incubating the surface biotinylated bacteria a with streptavidin modified magnetic nanoparticles to obtain the sound-magnetic dual-mode enhanced contrast agent; c. incubating the E. coli containing nanobubble gene after induced expression with biotinylated antibody to obtain surface biotinylated bacteria b, then incubating the surface biotinylated bacteria b with streptavidin modified magnetic nanoparticles to obtain the sound-magnetic dual-mode enhanced contrast agent.

[0014] Preferably, the induced expression comprises L-arabinose induced expression.

[0015] Preferably, the concentration of the L-arabinose is 0.3-0.7 g / mL; the volume of the L-arabinose is 1-3 mL; and the induced expression is performed by placing the E. coli in a 28-32 ℃, 150-170 rpm shaker for 20-24 hours.

[0016] Preferably, in a, the incubation reaction is performed at 100-140 rpm for 15-25 min; in b, the first incubation reaction is performed at 100-140 rpm for 0.5-1.5 h, and the second incubation reaction is performed at 100-140 rpm for 0.5-1.5 h; in c, the first reaction is performed at 100-140 rpm for 0.5-1.5 h, and the second reaction is performed at 100-140 rpm for 0.5-1.5 h; The biotinylated Sulfo-NHS is EZ-linkTM Sulfo-NHS-LC-biotin; and the biotinylated antibody is a biotin modified E. coli serotype O / K polyclonal antibody.

[0017] The application provides application of the above-mentioned acousto-magnetic dual-mode diagnosis and treatment agent or the acousto-magnetic dual-mode diagnosis and treatment agent prepared by the above-mentioned preparation method in preparation of a tumor diagnosis and / or treatment reagent.

[0018] Compared with the prior art, the application has the following beneficial effects: The application provides an acousto-magnetic dual-mode diagnosis and treatment agent, and the acousto-magnetic dual-mode diagnosis and treatment agent is a surface modification magnetic nanoparticle after induction expression of E. coli containing nanobubble genes. The E. coli in the acousto-magnetic dual-mode diagnosis and treatment agent generates nanobubbles inside, can be targeted and enriched in a tumor area, can generate a strong ultrasonic contrast signal, realizes ultrasonic contrast imaging, under external ultrasonic excitation, the nanobubbles have acoustic response characteristics, can vibrate and burst, thereby generating a mechanical effect, damaging target cells and tissues, and realizing sonoporation treatment of the tumor. Under the action of an external alternating magnetic field, a strong magnetic dynamic signal can be generated, the magnetic nanoparticles in the acousto-magnetic dual-mode diagnosis and treatment agent can mediate cell and tissue heating through a magnetic heating effect, and realize magnetic heating treatment. The magnetic nanoparticles in the acousto-magnetic dual-mode diagnosis and treatment agent can increase the acoustic activity of surrounding tissues, and have a synergistic effect with the ultrasonic contrast effect of the nanobubbles, further improving the imaging quality of the acousto-magnetic dual-mode enhanced diagnosis and treatment agent. The acousto-magnetic dual-mode diagnosis and treatment agent simultaneously has the magnetic nanoparticles and the nanobubbles, combines the advantages of two different imaging and treatment technologies, and provides more abundant diagnostic information and treatment strategies. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The working principle and application scene of the acousto-magnetic dual-mode diagnosis and treatment agent are shown in the figure. Figure 2 The process diagram of the surface modification of E. coli expressing nanobubble genes by using an amide coupling method to modify magnetic nanoparticles is shown in the figure. Figure 3 The process diagram of the surface modification of E. coli expressing nanobubble genes by using biotinylated Sulfo-NHS conjugated streptavidin modified magnetic nanoparticles is shown in the figure. Figure 4 The process diagram of the surface modification of E. coli expressing nanobubble genes by using biotinylated antibody conjugated streptavidin functionalized magnetic nanoparticles is shown in the figure. Figure 5Scanning electron micrographs of carboxyl magnetic nanoparticles, E. coli strains expressing nanobubbles or bacterial complexes modified with magnetic nanoparticles prepared in different examples, wherein (a) is a scanning electron micrograph of E. coli strains expressing nanobubbles prepared in Example 1; (b) is a scanning electron micrograph of carboxyl magnetic nanoparticles with a diameter of 100 nm described in Example 2; (c) is a scanning electron micrograph of streptavidin-modified magnetic nanoparticles with a diameter of 20 nm described in Example 3; (d) is a scanning electron micrograph of bacterial complexes modified with magnetic nanoparticles prepared in Example 2; (e) is a scanning electron micrograph of bacterial complexes modified with magnetic nanoparticles prepared in Example 3; (f) is a scanning electron micrograph of bacterial complexes modified with magnetic nanoparticles prepared in Example 4; Figure 6 Transmission electron micrographs of E. coli with or without ARG plasmid after induced expression, wherein (a) is a transmission electron micrograph of BL21 (AI) E. coli without ARG plasmid; (b) is a transmission electron micrograph of E. coli expressing nanobubbles prepared in Example 1 after induced expression; Figure 7 A gelatin phantom, the cross section of the phantom is a rectangle with a length of 60 mm and a width of 30 mm; the middle of the phantom is inlaid with brown bacteria modified with magnetic particles or magnetic particles, and the middle inlaid cross section is a square with a side length of 3 mm; Figure 8 Effects of bacterial phantom modified with magnetic particles on the contrast function of ultrasound imaging under different concentrations of E. coli expressing nanobubble genes, wherein (a) is a B-mode ultrasound image of the bacterial phantom modified with magnetic particles (the concentration of bacteria in the middle inlaid part is 1 x 10 9 mg / mL); (b) is a B-mode ultrasound image of the magnetic particle phantom (the concentration of magnetic particles is 10 mg / mL); (c) is a gray scale amplitude comparison of phantom 1 and phantom 2 under the same width and different depths; (d) is a B-mode ultrasound image of the bacterial phantom modified with magnetic particles (the concentration of bacteria is 4 x 10 9 mg / mL, and the corresponding concentration of magnetic particles is 10 mg / mL); (e) is a B-mode ultrasound image of the magnetic particle phantom (the concentration of magnetic particles is 10 mg / mL); (f) is a gray scale amplitude comparison of phantom 3 and phantom 4 under the same width and different depths; Figure 9 Effects of bacterial phantom modified with magnetic particles on the contrast function of magnetic resonance imaging under different concentrations of magnetic particles, wherein (a) is an excitation signal (alternating magnetic field frequency 250 Hz, cycle 10 cycles); (b) is the magnetic response vibration signal of the magnetic particle phantom (the concentration of magnetic particles is 10 mg / mL); (c) is the magnetic response vibration signal of the bacterial phantom modified with magnetic particles (the concentration of bacteria is 1 x 109 (d) is the magneto-mechanical vibration signal of the magnetic particle phantom (magnetic particle concentration is 10 mg / mL) under the excitation signal (alternating magnetic field frequency is 250 Hz, cycle is 10 cycles); (e) is the magneto-mechanical vibration signal of the magnetic particle phantom (magnetic particle concentration is 5 mg / mL); (f) is the magneto-mechanical vibration signal of the bacteria phantom (bacteria concentration is 1 x 10 9 (d) is the magneto-mechanical vibration signal of the magnetic particle phantom (magnetic particle concentration is 10 mg / mL) under the excitation signal (alternating magnetic field frequency is 250 Hz, cycle is 10 cycles); (e) is the magneto-mechanical vibration signal of the magnetic particle phantom (magnetic particle concentration is 5 mg / mL); (f) is the magneto-mechanical vibration signal of the bacteria phantom (bacteria concentration is 1 x 10 Figure 10 Fig. 1 is the magnetic verification result of the bacteria complex modified with the magnetic nanoparticles, (a) is the schematic diagram of the bacteria complex modified with the magnetic nanoparticles prepared in Example 2, (b) is the schematic diagram of the magnet placed on one side of the centrifuge tube containing the bacteria complex modified with the magnetic nanoparticles, (c) is the schematic diagram of the bacteria complex modified with the magnetic nanoparticles on one side after the magnet treatment, and a brown aggregation phenomenon appears; Figure 11 Fig. 2 is the verification result of the bacteria anaerobic characteristics, (a) is the verification result of the anaerobic characteristics before and after inoculating the BL21 (AI) E. coli without the ARG plasmid; (b) is the verification result of the anaerobic characteristics after inoculating the magnetic nanoparticles; (c) is the verification result of the anaerobic characteristics of the bacteria complex modified with the magnetic nanoparticles containing the ARG plasmid but not induced; Figure 12 Fig. 3 is the ultrasound magneto-mechanical imaging (red coding) and B-mode imaging (gray coding) result diagram of the bacteria complex modified with the magnetic nanoparticles; Figure 13 Fig. 4 is the result of plating the tumor and liver tissues taken from the mice after injecting the acoustic-magnetic dual-mode diagnosis and treatment agent for 1 day and 7 days, respectively, and the left column is the control group and the right column is the test group; Figure 14 Fig. 5 is that the nanobubble gas bag inside the bacteria of the acoustic-magnetic dual-mode diagnosis and treatment agent mediates the cell sonoporation under the external ultrasonic excitation, (a) is the tumor cell membrane before the sonoporation, (b) is the fluorescence diagram of the acoustic-magnetic dual-mode diagnosis and treatment agent, and (c) is the tumor cell membrane after the sonoporation; Figure 15 Fig. 6 is that the magnetic particles of the acoustic-magnetic dual-mode diagnosis and treatment agent mediate the tumor heating of the mice under the external alternating magnetic field excitation. DETAILED DESCRIPTION

[0020] The present application provides an acoustic-magnetic dual-mode diagnosis and treatment agent, which is the surface modified magnetic nanoparticle after the induced expression of the nanobubble gas bag gene containing E. coli, and the nucleotide sequence of the nanobubble gas bag gene is NCBI: KC601845.1.

[0021] In the present application, the nucleotide sequence of the nanobubble gene is the full-length sequence shown in NCBI: KC601845.1, i.e., the nucleotide sequence shown in positions 1-18448.

[0022] In the present application, the E. coli containing the nanobubble gene in the acoustic-magnetic dual-mode diagnosis and treatment agent has active targeting property to the tumor hypoxic region as a diagnosis and treatment agent carrier, the bacteria contain nanobubbles inside, and have acoustic response property; the E. coli is surface-modified with magnetic nanoparticles, and has magnetic response property. In acoustic-magnetic dual-mode imaging, firstly, the nanobubbles expressed inside the bacteria can produce enhanced tumor ultrasonic echo signals on the real-time ultrasonic contrast monitoring image; secondly, under the action of an external changing magnetic field, the magnetic nanoparticles on the surface of the bacteria can produce enhanced tumor magnetic vibration imaging effect. In acoustic-magnetic combined treatment, firstly, when ultrasonic excitation is performed, the nanobubbles expressed inside the bacteria can produce sonoporation effect to kill cells; secondly, when alternating magnetic excitation is performed, the magnetic nanoparticles on the surface of the bacteria can produce magnetic heating effect to kill cells. The acoustic-magnetic dual-mode enhanced diagnosis and treatment agent can realize more efficient tumor diagnosis and / or treatment.

[0023] In the present application, the E. coli is preferably an E. coli strain with T7 RNA polymerase and for protein expression driven by a T7 promoter, and is preferably an E. coli BL21 strain, such as E. coli BL21 (AI) or E. coli BL21 (DE3).

[0024] In the present application, the construction method of the E. coli containing the nanobubble gene comprises the following steps: The expression vector containing the nanobubble gene is transformed into the E. coli, and the E. coli containing the nanobubble gene is obtained.

[0025] In the present application, the expression vector containing the nanobubble gene preferably comprises a plasmid with a T7 promoter, further preferably a pBAD plasmid or a pET plasmid, and more preferably a pBAD33. The construction method of the expression vector containing the nanobubble gene comprises inserting a nanobubble gene (also referred to as an ARG gene) between XbaI and SmaI sites in the backbone plasmid pBAD33. The ARG gene is a nanobubble gene from Serratia sp. 39006 (the sequence of the nanobubble gene is shown in the full-length nucleotide sequence in positions 1-18448 of NCBI: KC601845.1).

[0026] In the present application, the expression vector containing nanobubble gene is introduced into E. coli, and the E. coli can generate double-cone-shaped nanobubbles with the size of about 45 nm in width and 200 nm in length in its interior through L-arabinose induction expression. The main principle of the imaging of the acoustic-magnetic dual-mode diagnosis and treatment agent of the present application is to utilize the acoustic impedance difference between the nanobubbles and the surrounding tissues to significantly enhance the ultrasonic echo signal, thereby improving the resolution, sensitivity and specificity of the ultrasonic imaging.

[0027] Preferably, the surface modification of the magnetic nanoparticle after the induction expression of the E. coli containing nanobubble gene includes any one of the following modes: (1) the carboxyl magnetic nanoparticle is modified on the surface of the E. coli containing nanobubble gene after the induction expression through amide coupling method; (2) the Sulfo-NHS biotinization is modified on the surface of the E. coli containing nanobubble gene after the induction expression, and the magnetic nanoparticle modified by streptavidin is connected; (3) the antibody biotinization is modified on the surface of the E. coli containing nanobubble gene after the induction expression, and the magnetic nanoparticle modified by streptavidin is connected.

[0028] The present application provides a preparation method of the acoustic-magnetic dual-mode enhanced contrast agent, which is prepared by any one of the following methods a~c: a. the carboxyl magnetic nanoparticle, EDC and Sulfo-NHS are mixed to obtain the activated magnetic nanoparticle; then the E. coli containing nanobubble gene after the induction expression is incubated with the activated magnetic nanoparticle to obtain the same; b. the E. coli containing nanobubble gene after the induction expression is incubated with the Sulfo-NHS biotinization to obtain the surface biotinization bacteria a, and then the surface biotinization bacteria a is incubated with the magnetic nanoparticle modified by streptavidin to obtain the same; c. the E. coli containing nanobubble gene after the induction expression is incubated with the antibody biotinization to obtain the surface biotinization bacteria b, and then the surface biotinization bacteria b is incubated with the magnetic nanoparticle modified by streptavidin to obtain the same.

[0029] In the present application, the induced expression includes the use of L-arabinose to induce expression, wherein the concentration of the L-arabinose is 0.3-0.7 g / mL, further preferably 0.4-0.6 g / mL, and more preferably 0.4, 0.5 or 0.6 g / mL; the volume of the L-arabinose is 1-3 mL, further preferably 1.5-2.5 mL, and more preferably 2 mL; and the mode of the induced expression is incubation at 28-32°C in a 150-170 rpm shaker for 20-24 hours, and as a preferred embodiment, the mode of the induced expression is incubation at 30°C in a 160 rpm shaker for 22 hours. The bacterial complex of the E. coli induced expression containing the nanobubble gene and the surface modified magnetic nanoparticle can be successfully prepared by the above preparation method. In the above mode a, the E. coli cell wall components contain amino groups, which can react with the carboxyl groups on the surface of the carboxylated magnetic nanoparticles under the action of the condensing agent (EDC, Sulfo-NHS) to form a firm amide covalent bond, thereby achieving the modification of the magnetic nanoparticles on the surface of the bacteria. The mode of the incubation reaction is 100-140 rpm for 15-25 min, further preferably 110-130 rpm for 17-23 h, and more preferably 120 rpm for 20 min. After the incubation, the E. coli containing the nanobubble gene can be coupled with the activated magnetic nanoparticles through amide coupling, and the activity of the target bacteria can also be maintained. The carboxylated magnetic nanoparticles are magnetic particles with carboxyl groups on the surface, and the carboxylated magnetic nanoparticles, carboxylated ferroferric oxide nanoparticles, carboxylated PEG modified magnetic nanoparticles, carboxylated dextran modified magnetic nanoparticles or carboxylated dextran modified ferroferric oxide nanoparticles can be used. In the above mode b, the E. coli containing the nanobubble gene after induced expression is co-incubated with biotinylated Sulfo-NHS to achieve the biotinylation on the surface of the bacteria. The streptavidin functionalized magnetic nanoparticles can bind to the biotin on the surface of the bacteria to achieve the effective modification of the magnetic nanoparticles. The mode of the first incubation reaction is preferably 100-140 rpm for 0.5-1.5 h, further preferably 115-135 rpm for 0.5-1.5 h, and more preferably 120 rpm for 1 h. The mode of the second incubation reaction is 100-140 rpm for 0.5-1.5 h, further preferably 115-135 rpm for 0.5-1.5 h, and more preferably 120 rpm for 1 h. In the above mode c, the E. coli containing the nanobubble gene has O antigen (bacterial antigen), K antigen (surface antigen) and other antigens on the surface, which can bind to the corresponding biotin modified antibodies to achieve the biotinylation on the surface of the bacteria, and the streptavidin modified magnetic nanoparticles can bind to the biotin on the surface of the bacteria to achieve the effective modification of the magnetic nanoparticles.The first reaction is incubated at 100-140 rpm for 0.5-1.5 h, further preferably at 115-135 rpm for 0.5-1.5 h, more preferably at 120 rpm for 1 h, and the second reaction is incubated at 100-140 rpm for 0.5-1.5 h, further preferably at 115-135 rpm for 0.5-1.5 h, more preferably at 120 rpm for 1 h.

[0030] In the present application, the biotinylated Sulfo-NHS is EZ-linkTM Sulfo-NHS-LC-biotin, and the biotinylated antibody is a biotin-modified E. coli serotype O / K polyclonal antibody. The present application does not have special limitations on EZ-linkTM Sulfo-NHS-LC-biotin and biotin-modified E. coli serotype O / K polyclonal antibody, and commercially available products known in the art can be used.

[0031] As shown in Figure 1 The acoustic-magnetic dual-mode enhanced diagnosis and treatment agent of the present application mainly includes acoustic response characteristics and magnetic response characteristics. The two response characteristics can be applied to imaging and treatment. The ultrasonic imaging acquisition instrument is responsible for acquiring the signal received by the ultrasonic transducer, and then realizing ultrasonic contrast imaging, ultrasonic elastography and ultrasonic magnetic dynamic imaging; secondly, it is responsible for outputting ultrasonic excitation signals for inducing sonophoresis treatment. In addition, the ultrasonic imaging instrument can output a trigger signal, and the trigger signal generator outputs a small electrical signal, which is amplified by a power amplifier to drive a magnetic excitation coil to output an alternating magnetic field for inducing magnetic dynamic imaging and magnetic heat treatment.

[0032] The carrier of the acoustic-magnetic dual-mode diagnosis and treatment agent of the present application is bacteria, and the enrichment mode of the diagnosis and treatment agent has two kinds, the first kind is active movement of bacteria, such as E. coli as a carrier, which can move to anoxic area; the second kind is passive attraction of magnet, such as passive attraction of diagnosis and treatment agent to the treatment area by the external placed magnetic field, so as to effectively target the tumor area and achieve the purpose of precise treatment of tumor.

[0033] When the acoustic response characteristics of the diagnosis and treatment agent are applied to imaging, the bacteria contain nanobubbles inside, so they have echo enhancement characteristics. This echo enhancement characteristic can be collected by an ultrasonic imaging instrument to realize ultrasonic contrast enhancement imaging, thereby achieving the effect of diagnosing tumors.

[0034] The magnetic response characteristics of the diagnosis and treatment agent are applied to imaging: mainly including two parts, the first part is that the magnetic nanoparticles on the surface of the E. coli are affected by the changing magnetic field, causing the E. coli to produce magnetic vibration, and the vibration information (vibration displacement size, vibration spatial position) of the E. coli is detected by using the ultrafast ultrasound imaging mode, so that the concentration information and spatial distribution information of the E. coli in the tumor area can be obtained. The second part is that when the E. coli and the magnetic nanoparticles vibrate, the shear wave propagation in the surrounding tissue is triggered. The propagation of the shear wave is detected by using the elastic ultrasound imaging mode, so that the elasticity information of the tumor tissue around the E. coli and the magnetic nanoparticles can be obtained.

[0035] The acoustic response characteristics of the acoustic-magnetic dual-mode diagnosis and treatment agent are applied to treatment: the E. coli contains a nano gas bag inside. When excited by ultrasound, the nano gas bag will vibrate and burst, thereby producing a mechanical effect to destroy the structure of the bacteria, release the magnetic nanoparticles and kill the nearby cells. The parameters of the ultrasound are 1-10 MHz center frequency, 0.8-2.0 MPa negative acoustic pressure, 1%-30% duty cycle, 1 Hz-1 kHz pulse repetition frequency, and 4-6 cycles.

[0036] The magnetic response characteristics of the acoustic-magnetic dual-mode diagnosis and treatment agent are applied to treatment: the bacteria carry magnetic particles outside. In the two states of not releasing and releasing the magnetic particles, the magnetic heat effect can be induced by an external alternating magnetic field, thereby increasing the temperature, denaturing the protein and killing the nearby cells. When the diagnosis and treatment agent in the application is used for treatment, the acoustic-magnetic combined excitation can kill the target cells in double effect.

[0037] The application provides an application of the above-mentioned acoustic-magnetic dual-mode diagnosis and treatment agent or the acoustic-magnetic dual-mode diagnosis and treatment agent prepared by the preparation method in the preparation of a tumor diagnosis and / or treatment reagent.

[0038] In the application, the tumor is breast cancer.

[0039] In the application, all raw material components are commercially available goods well known to those skilled in the art without special instructions.

[0040] The technical solutions provided by the application will be described in detail below in combination with embodiments, but they should not be understood as limitations to the protection scope of the application.

[0041] In the following embodiments, the main culture medium and related materials are as follows: LB liquid medium: 5 g of LB liquid medium dry powder is weighed and dissolved in 200 mL of pure water, and after complete dissolution, 1 mol / L NaOH solution is used to adjust the pH value to 7.0-7.2. The culture medium is placed in a high-pressure steam sterilization pot for liquid sterilization. After sterilization is completed, the culture medium is sealed and stored at 4℃ for standby.

[0042] 50 mg / mL chloramphenicol stock solution: 500 mg of chloramphenicol powder was weighed and dissolved in an appropriate amount of absolute ethanol, and then the absolute ethanol was used to make up to 10 mL. Under sterile conditions, a 22-μm microporous filter was used for filtration and aliquotting. After sealing, it was stored at -20°C in the dark.

[0043] LB solid selective medium: 5 g of LB solid medium dry powder was weighed and dissolved in 200 mL of pure water. After complete dissolution, 1 mol / L NaOH solution was used to adjust the pH to 7.0-7.2. The medium was placed in a high-pressure steam sterilization pot for liquid sterilization. After sterilization was completed, when the medium temperature decreased to 50°C, 100 μL of 50 mg / mL chloramphenicol stock solution was added under sterile conditions, so that the final antibiotic concentration was 25 μg / mL. Then the medium was poured into a plate, and after it solidified, the plate was stored at 4°C.

[0044] 0.4 g / mL glucose stock solution: 4 g of glucose was weighed and dissolved in an appropriate amount of pure water, and then the pure water was used to make up to 10 mL. Under sterile conditions, a 22-μm microporous filter was used for filtration and aliquotting. After sealing, it was stored at 4°C.

[0045] Preparation of LB liquid medium 1: In 5 mL of LB liquid medium, 2.5 μL of 50 g / mL chloramphenicol stock solution and 125 μL of 0.4 g / mL glucose stock solution were added to obtain LB liquid medium 1.

[0046] Preparation of LB liquid medium 2: In 200 mL of LB liquid medium, 100 μL of 50 mg / mL chloramphenicol stock solution and 1 mL of 0.4 g / mL glucose stock solution were added to obtain LB liquid medium 2.

[0047] 50% glycerol solution: 20 mL of 1×PBS solution was added to 20 mL of pure glycerol, and then mixed uniformly and placed in a high-pressure steam sterilization pot for liquid sterilization. After sterilization was completed, it was sealed and stored at 4°C.

[0048] 0.5 g / mL L-arabinose stock solution: 5 g of L-arabinose was dissolved in an appropriate amount of pure water, and then the pure water was used to make up to 10 mL. Under sterile conditions, a 22-μm microporous filter was used for filtration and aliquotting. After sealing, it was stored at 4°C.

[0049] BL21(AI) competent cells: purchased from Thermo Scientific, catalog number C607003.

[0050] Example 1 Construction of E. coli strain expressing nanocell gene (1) Construction of plasmid carrying nanoscale airbag gene (referred to as ARG plasmid): the ARG gene is cloned into the XbaI and Smal endonuclease of the backbone plasmid pBAD33 to obtain the ARG plasmid; the nucleotide sequence of the nanoscale airbag gene is the nucleotide sequence shown in positions 1-18448 in NCBI: KC601845.1.

[0051] (2) Take 100 μL of BL21 (AI) competent cells melted on ice, add 100 ng of ARG plasmid, mix gently, and then place on ice for 25 min, heat shock in a 42°C water bath for 45 s, quickly return to ice and stand for 2 min, to obtain the transformed competent cells; (3) Add 700 μL of LB liquid medium to the transformed competent cells, mix, and then place in a 37°C, 200 rpm shaker for 60 min, centrifuge at 5000 rpm for 1 min at 24°C, remove 600 μL of supernatant, take about 100 μL of supernatant and resuspend the bacteria by gently blowing, and evenly coat on the LB solid selection medium, invert the plate, and place in a 37°C incubator for 12 h, select 2 single colonies from the plate, inoculate in 5 mL of LB liquid medium 1, and place in a 37°C, 200 rpm shaker for single colony culture for 12 h, to obtain the bacterial culture; (4) Inoculate 2 mL of the above bacterial culture into 200 mL of LB liquid medium 2, and place in a 37°C, 200 rpm shaker for 6 h of expansion culture, identify and select positive expression of E. coli, to obtain E. coli expressing nanoscale airbag gene.

[0052] In a 2 mL centrifuge tube, add 0.5 mL of E. coli expressing nanoscale airbag gene and 0.5 mL of 50% glycerol solution, mix evenly, seal, and store at -80°C, to obtain the glycerol bacteria carrying ARG plasmid.

[0053] Example 2 2.1 Reagents and related materials: Carboxyl magnetic nanoparticles with a diameter of 100 nm were purchased from Nanjing Dongna Biological Technology Co., Ltd. (product number Mag9000), with a concentration of 10 mg / mL and a surface carboxyl content of about 350 nmol / mg.

[0054] Activation buffer: add 0.0292 g of sodium chloride to 1 mL of 0.1 mol / L MES solution to make the final concentration of sodium chloride 0.5 M (mol / L), and the pH is 6.0.

[0055] The 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is referred to as EDC, and is purchased from Thermo Scientific (product number PG82079).

[0056] The N-hydroxysulfosuccinimide is referred to as Sulfo-NHS, which is purchased from Thermo Scientific (item number A39269).

[0057] 2.2 Modification of ferroferric oxide nanoparticles on the surface of E. coli expressing nanomembrane genes by amide coupling method, the steps are as follows: (1) 1 mL of glycerol bacteria carrying ARG plasmid prepared in Example 1 was inoculated into 200 mL of LB liquid medium 2, and cultured at 37°C, 200 rpm on a shaker for 1.5 h to OD 600 =0.5, 2 mL of L-arabinose stock solution with a concentration of 0.5 g / mL was added, and it was cultured at 30°C, 160 rpm on a shaker for 22 hours to obtain E. coli induced to express nanomembranes; (2) The E. coli induced to express nanomembranes was centrifuged at 300 rcf for 10 min at 4°C, the first precipitate was discarded, the culture medium in the supernatant was sucked out by inserting a syringe at the bottom of the liquid surface, and the bacteria at the liquid surface were reserved. 40 mL of 1×PBS solution was added, and the second precipitate was discarded by centrifugation at 300 rcf for 10 min at 4°C. The 1×PBS solution in the supernatant was sucked out by inserting a syringe at the bottom of the liquid surface, and the first washed bacteria were obtained; (3) 40 mL of 1×PBS solution was added, and step (2) was repeated 4 times for washing. The washed bacteria were resuspended in an appropriate amount of 1×PBS solution, and the concentration of the E. coli induced to express nanomembranes solution was adjusted to 1×10 9 / mL; (4) 500 μL of carboxyl magnetic nanoparticles (mNPs) were added to a 2 mL centrifuge tube, and the magnetic nanoparticles were magnetically adsorbed using a magnetic stand with a magnetic strength of 420 mT at room temperature (22-25°C). After discarding the supernatant (unadsorbed solution), 1.8 mL of activation buffer was added to resuspend the magnetic nanoparticles in the centrifuge tube. 10 mg of Sulfo-NHS was weighed and added to the centrifuge tube for shaking and dissolving. Then 20 mg of EDC was added to the centrifuge tube for shaking and dissolving. The centrifuge tube was placed in a plate shaker and shaken at 120 rpm at room temperature for 10 min to obtain activated magnetic nanoparticles.

[0058] (5) The activated magnetic nanoparticles were magnetically adsorbed using a magnetic stand with a magnetic strength of 420 mT at room temperature. After discarding the supernatant, 1 mL of 1×PBS solution was added and mixed evenly, and the adsorption and separation were continued. The supernatant was discarded. (6) Add 1 mL of 1xPBS solution, repeat step (5) for 3 times, then add 500 μL of 1xPBS solution to the obtained washed magnetic nanoparticles and mix well by blowing, and then disperse the magnetic nanoparticles by ultrasonic wave to obtain a solution of washed carboxyl magnetic nanoparticles.

[0059] (7) In a 2 mL centrifuge tube, add 1 mL of 1x10 9 9 / mL of E. coli solution for expressing nanomembranosome obtained in step (3) and 500 μL of the carboxyl magnetic nanoparticle solution prepared in step (6), and place the centrifuge tube in a flat-bed shaker and incubate at room temperature at 120 rpm for 20 min to obtain a bacterial complex after reaction.

[0060] (8) Centrifuge the bacterial complex after reaction at 4°C at 300 rcf for 10 min, discard the third precipitate, and then insert a syringe into the bottom of the liquid surface to remove the solution in the supernatant, retain the bacteria on the liquid surface, add 1.5 mL of 1xPBS solution, mix well, and continue centrifugation, discard the fourth precipitate, and then insert a syringe into the bottom of the liquid surface to remove the 1xPBS solution in the supernatant; (9) Add 1.5 mL of 1xPBS solution, repeat step (8) for 4 times for washing, add 1.5 mL of 1xPBS solution to the obtained washed bacterial complex to resuspend, and obtain a resuspended bacterial complex; (10) At room temperature, use a magnetic stand with a magnetic force of 420 mT to magnetically adsorb the resuspended bacterial complex, discard the solution containing bacteria that are not successfully modified (unadsorbed solution), add 1 mL of 1xPBS solution, mix well, and continue to adsorb and separate, and discard the supernatant (unadsorbed solution); (11) Add 1 mL of 1xPBS solution, repeat step (10) for 3 times to obtain a bacterial complex modified with magnetic nanoparticles on the surface, which is also referred to as a magneto-acoustic dual-mode diagnostic and therapeutic agent.

[0061] The process diagram of modifying magnetic nanoparticles on the surface of E. coli expressing nanomembranosome genes by amide coupling method is shown in Figure 2 .

[0062] Example 3 3.1 Reagents and Related Materials: The streptavidin-modified magnetic nanoparticles used in this example have a diameter of 20 nm and are purchased from Nanjing Dongna Biological Technology Co., Ltd. (product code Mag3301). The concentration is 1 mg / mL, and the surface streptavidin content is about 60 μg / mg.

[0063] 10 mM EZ-link TM Sulfo-NHS-LC-Biotin solution: 2.2 mg of EZ-link TMThe Sulfo-NHS-LC-Biotin powder was dissolved in 500 µL of pure water to obtain the EZ-link. TM Sulfo-NHS-LC-Biotin was purchased from Thermo, catalog number 21335.

[0064] 3.2 Biotinylated Sulfo-NHS was modified on the surface of E. coli expressing the nano-airbag gene, and streptavidin-functionalized magnetic nanoparticles were combined to achieve the modification of magnetic nanoparticles on the surface of E. coli expressing the nano-airbag gene. The steps are as follows: (1) The 1×10 prepared in step (3) of Example 2 9 1 mL of E. coli solution containing 10 mM EZ-link cells / mL and 200 μL of 10 mM EZ-link. TM Sulfo-NHS-LC-Biotin solution was used to incubate centrifuge tubes in a plate shaker at 120 rpm for 1 h at room temperature to obtain biotinylated bacteria. (2) Centrifuge the surface biotinylated bacteria at 300 rcf for 10 min at room temperature, discard precipitate a, insert a syringe into the bottom of the liquid surface to aspirate the solution in the supernatant, retain the bacteria on the liquid surface, add 1.5 mL of 1×PBS solution, mix well and continue centrifuging, discard precipitate b, insert a syringe into the bottom of the liquid surface to aspirate the 1×PBS solution in the supernatant. (3) Add 1.5 mL of 1×PBS solution, repeat step (2) 3 times, and then add 1 mL of 1×PBS solution to obtain the washed bacterial solution; (4) Add 100 μL of streptavidin-modified magnetic nanoparticles to the washed bacterial solution. Place the centrifuge tube in a plate shaker and incubate at 120 rpm for 1 h at room temperature to obtain the incubated bacterial complex; (5) Centrifuge the incubated bacterial complex at 300 rcf for 10 min at 4 °C, discard the precipitate c, insert the syringe to the bottom of the liquid surface to aspirate the 1×PBS solution in the supernatant, retain the bacteria on the liquid surface, add 1.5 mL of 1×PBS solution, mix well and continue centrifuging, discard the precipitate d, insert the syringe to the bottom of the liquid surface to aspirate the 1×PBS solution in the supernatant. (6) Add 1.5 mL of 1×PBS solution and repeat step (5) 4 times to wash. Add 1.5 mL of 1×PBS solution to the washed bacterial complex and resuspend it to obtain the resuspended bacterial complex. (7) At room temperature, the bacterial complex was magnetically adsorbed and resuspended using a magnetic rack with a magnetic strength of 420 mT. The solution containing unmodified bacteria (unadsorbed solution) was discarded. 1 mL of 1×PBS solution was added, mixed, and separation was continued. The supernatant (unadsorbed solution) was discarded. (8) Add 1 mL of 1xPBS solution, repeat step (7) for 3 times, to obtain the bacteria complex modified with magnetic nanoparticles on the surface, also known as the magneto-acoustic dual-mode theranostic agent.

[0065] The process chart for modifying biotinylated Sulfo-NHS-streptavidin modified magnetic nanoparticles on the surface of E. coli expressing nanomembrane vesicle genes is shown in Figure 3 .

[0066] Example 4 4.1 Reagents and related materials: The streptavidin modified magnetic nanoparticles used in this example have a diameter of 20 nm, purchased from Nanjing Dongna Biotechnology Co., Ltd. (product code Mag3301), with a concentration of 1 mg / mL and a surface streptavidin content of about 60 μg / mg.

[0067] Biotin modified E. coli serotype O / K polyclonal antibody was purchased from Invitrogen (product code PA1-73031) with a concentration of 5 mg / mL.

[0068] 4.2 Modify biotinylated antibody on the surface of E. coli expressing nanomembrane vesicle genes, and combine streptavidin functionalized magnetic nanoparticles to achieve the modification of magnetic nanoparticles on the surface of E. coli expressing nanomembrane vesicle genes, as follows: (1) 1 mL of 1x10 9 E. coli solution induced to express nanomembrane vesicles at a concentration of 1x10 (2) Centrifuge the biotinylated surface modified bacteria at 300 rcf for 10 min at room temperature, discard the precipitate 1, insert a syringe into the bottom of the liquid surface to remove the solution in the supernatant, add 1.5 mL of 1xPBS solution, mix well and continue centrifugation, discard the precipitate 2, insert a syringe into the bottom of the liquid surface to remove the 1xPBS solution in the supernatant; (3) Add 1.5 mL of 1xPBS solution, repeat step (2) for 3 times, then add 1 mL of 1xPBS solution, to obtain the washed bacteria solution; (4) Add 100 μL of streptavidin modified magnetic nanoparticles to the washed bacteria solution. Place the centrifuge tube in a plate shaker and incubate at 120 rpm for 1 h at room temperature to obtain the bacteria complex; (5) centrifuge the bacteria complex at 300rcf for 10min at 4℃, keep the bacteria in the liquid surface, discard the precipitate 3, inject a syringe into the bottom of the liquid surface to discard the 1xPBS solution in the supernatant, add 1.5mL of 1xPBS solution, mix and continue centrifugation, discard the precipitate 4, inject a syringe into the bottom of the liquid surface to discard the 1xPBS solution in the supernatant; (6) add 1.5mL of 1xPBS solution, repeat step (5) 4 times to wash, add 1.5mL of 1xPBS solution to resuspend the obtained washed bacteria complex to obtain the resuspended bacteria complex; (7) use a magnetic stand to magnetically attract the resuspended bacteria complex at room temperature with a magnetic strength of 420mT, discard the solution containing the bacteria which are not successfully modified (unattracted solution), add 1mL of 1xPBS solution to mix and continue separation, discard the supernatant (unattracted solution); (8) add 1mL of 1xPBS solution to repeat step (7) 3 times to obtain the bacteria complex which is modified with the magnetic nanoparticles on the surface, also known as the acoustic-magnetic dual-mode diagnosis and treatment agent.

[0069] The process diagram for realizing the modification of the bacteria surface with the magnetic nanoparticles by the biotinylated antibody binding streptavidin method is shown in Figure 4 .

[0070] Example 5 The difference between this example and example 2 is that in step (4) of this example, 3mL of carboxyl magnetic nanoparticles are added in the centrifuge tube, and the remaining steps of step (4) of this example are the same as those of example 2; in step (7) of this example, 1mL of the 1.2x10 10 9

[0071] Example 6 The difference between this example and example 2 is that in step (4) of this example, 3mL of carboxyl magnetic nanoparticles are added in the 2mL centrifuge tube, and the remaining steps of step (4) of this example are the same as those of example 2; in step (7) of this example, 3x10 9 ​​Example 7 9 Example 8

[0072] Example 7 The difference between this example and Example 2 is that in step (4) of this example, 1.5 mL of carboxyl magnetic nanoparticles are added into a 2 mL centrifuge tube, and the remaining steps of step (4) of this example are the same as those of Example 2; in step (7) of this example, 3 x 10 9 Example 7 9 Example 8

[0073] Example 8 8.1 The E. coli strain expressing nanomembrane sac prepared in Example 1, the carboxyl magnetic nanoparticles described in Example 2, the streptavidin-modified magnetic nanoparticles described in Example 3, and the bacterial complex modified with magnetic nanoparticles prepared in Examples 2-4 are observed by scanning electron microscopy and photographed.

[0074] Figure 5 The results show that the magnetic nanoparticles can be modified on the surface of the E. coli strain expressing nanomembrane sac by using the amide coupling method, the biotinylated Sulfo-NHS binding streptavidin method, and the biotinylated antibody binding streptavidin method. The "amide coupling method" can be used to modify large-sized magnetic nanoparticles (≥ 100 nm) on the surface of the bacteria; the "biotinylated Sulfo-NHS binding streptavidin method" and the "biotinylated antibody binding streptavidin method" are suitable for modifying small-sized magnetic nanoparticles (20-50 nm) on the surface of the bacteria.

[0075] 8.2 In vitro induction of expression of nanomembrane sac In 1 mL of the suspension of the E. coli expressing nanobubble gene prepared in Example 1, 10 μL of L-arabinose stock solution with a concentration of 0.5 g / mL was added, mixed, and the resulting mixture was placed in a 30°C, 180 rpm shaker for induction for 22 h to obtain the BL21 (AI) bacteria after induction expression. Meanwhile, the E. coli BL21 (AI) without the ARG plasmid was mixed with 10 μL of pure water, and the resulting mixture was placed in a 30°C, 180 rpm shaker for 22 h to obtain the BL21 (AI) bacteria without the ARG plasmid (control group).

[0076] The BL21 (AI) bacteria after induction expression of nanobubbles and the BL21 (AI) bacteria without the ARG plasmid were observed and photographed by transmission electron microscopy.

[0077] Figure 6 The results show that, compared with the control group, the E. coli expressing nanobubble genes can generate double-cone-shaped nanobubbles with a size of about 45 nm wide and 200 nm long inside after induction expression, indicating that the E. coli expressing nanobubble genes is successfully prepared in Example 1.

[0078] 8.3 Phantom experiment a, gelatin phantom making, Figure 7 is a gelatin phantom.

[0079] a-1, related materials Preparation of gelatin solution 1: In a beaker, add 200 mL of deionized water, 20 g of pigskin gelatin powder (purchased from ABCONE, product number G32194), and 6.6 g of corn starch, and stir until uniform. Cover the beaker with plastic wrap and leave a small gap for air exchange. Heat in a microwave oven at a power of 700 W (if signs of boiling are observed, immediately stop heating to prevent overflow, and stir). Repeat the heating step until the solution changes from milky white to clear and transparent, without bubbles and milky white flocculation. After stopping heating, place the beaker on a magnetic stirrer at 200 rpm and control the temperature at 38°C for standby.

[0080] Preparation of gelatin solution 2: In a beaker, add 100 mL of deionized water, 20 g of pigskin gelatin powder, and 6.6 g of corn starch, and stir until uniform. Cover the beaker with plastic wrap and leave a small gap for air exchange. Heat in a microwave oven at a power of 700 W (if signs of boiling are observed, immediately stop heating to prevent overflow, and stir). Repeat the heating step until the solution changes from milky white to clear and transparent, without bubbles and milky white flocculation. After stopping heating, place the beaker on a magnetic stirrer at 200 rpm and control the temperature at 40°C for standby.

[0081] a-2, pour the gelatin solution 1 into the mold (liquid level 3mm), remove the bubbles, and store at 4℃ for 15min to solidify, to obtain a solidified phantom; a-3, take 1mL of the gelatin solution 2 and 1mL of the bacterial complex solution modified with magnetic nanoparticles prepared in examples 5-7, or take 1mL of the gelatin solution 2 and 1mL of the carboxyl magnetic nanoparticles with a concentration of 10mg / mL or 20mg / mL, mix them uniformly at 40℃ water bath, to obtain each mixed solution, pour the mixed solution into the mold, and store at 4℃ for 20min to solidify. Cut the solidified material into a cube with a size of 3mm×3mm×3mm, and place it at the center of the solidified phantom obtained in step a-2, to obtain a mold containing an embedded block; a-4, cool the gelatin solution 1 to 31℃, pour it into the mold containing the embedded block obtained in step a-3, remove the bubbles, and store at 4℃ for 30min to solidify, to obtain a bacterial phantom modified with magnetic particles or a magnetic particle phantom.

[0082] b, perform ultrasonic B-mode imaging on the gelatin phantom.

[0083] Figure 8 (a) in figure (a) is a bacterial phantom modified with magnetic particles (bacterial concentration is 1×10 9 mg / mL, magnetic particle concentration is 10mg / mL), denoted as phantom 1; Figure 8 (b) in figure (b) is a magnetic particle phantom (magnetic particle concentration is 10mg / mL), denoted as phantom 2; Figure 8 (d) in figure (d) is a bacterial phantom modified with magnetic particles (bacterial concentration is 4×10 9 mg / mL, magnetic particle concentration is 10mg / mL), denoted as phantom 3; Figure 8 (e) in figure (e) is a magnetic particle phantom (magnetic particle concentration is 10mg / mL), denoted as phantom 4; Figure 8 (c) in figure (c) is a gray scale amplitude contrast of phantom 1 and phantom 2 at the same width and different depths; Figure 8 (f) in figure (f) is a gray scale amplitude contrast of phantom 3 and phantom 4 at the same width and different depths.

[0084] Figure 8 The results show that the single magnetic nanoparticle region of phantom 2 and phantom 4 has no obvious ultrasonic signal enhancement, the magnetic nanoparticle modified bacterial region of phantom 1 and phantom 3 has obvious ultrasonic signal enhancement (as indicated by the black arrow), and the ultrasonic signal of phantom 3 with higher bacterial concentration is stronger than that of phantom 1. It is proved that the E. coli expressing nanobubble sacs has the ability to enhance the ultrasonic echo signal, and the higher the concentration of the E. coli expressing nanobubble sacs, the stronger the ultrasonic echo signal. The above results prove the ultrasonic imaging contrast enhancement function of the present application.

[0085] c. Extract the vibration signal of the gel phantom under the magnetic field.

[0086] Figure 9 (a) and (d) in FIG. 10 are the vibration signals of the gel phantom under the magnetic field, wherein (a) and (d) are the vibration signals of the gel phantom under the magnetic field, and (a) and (d) are the vibration signals of the gel phantom under the magnetic field.

[0087] Figure 9 (b) in FIG. 10 is a magnetic particle phantom (the concentration of the magnetic particles is 10 mg / mL), which is referred to as phantom 5. Figure 9 (c) in FIG. 10 is a bacteria phantom modified with magnetic particles (the concentration of the bacteria is 1×10 9 mg / mL, and the concentration of the magnetic particles is 10 mg / mL), which is referred to as phantom 6. Figure 9 (e) in FIG. 10 is a magnetic particle phantom (the concentration of the magnetic particles is 5 mg / mL), which is referred to as phantom 7. Figure 9 (f) in FIG. 10 is a bacteria phantom modified with magnetic particles (the concentration of the bacteria is 1×10 9 mg / mL, and the concentration of the magnetic particles is 5 mg / mL), which is referred to as phantom 8.

[0088] Figure 9 The results in Table 1 show that the magnetic dynamic response vibration signal of the bacteria phantom 6 modified with magnetic nanoparticles (10 mg / mL) is stronger than that of the phantom 5, and the average amplitude is increased by 39.94%. The magnetic dynamic response vibration signal of the bacteria phantom 8 modified with magnetic nanoparticles (5 mg / mL) is stronger than that of the phantom 7, and the average amplitude is increased by 60.85%. It is proved that the bacteria phantom modified with magnetic particles has the ability to enhance the magnetic dynamic signal, and the higher the concentration of the magnetic particles, the stronger the magnetic dynamic signal. The above results prove the contrast enhancement function of the magnetic dynamic imaging of the present application.

[0089] 8.4 Verification of the function of the magneto-audio dual-mode diagnosis and treatment agent a. Magnetic verification The bacteria complex modified with magnetic nanoparticles on the surface prepared in Example 2 is placed on one side of a centrifugal tube containing the bacteria complex, and a photograph is taken.

[0090] Figure 10 The results in Table 2 show that the bacteria complex modified with magnetic nanoparticles on the surface will quickly appear brown aggregation on one side, and has magnetism.

[0091] b. Verification of hypoxic characteristics Preparation of semi-solid medium: 17.5 g of LB liquid medium dry powder and 3.5 g of agar powder were weighed into 700 mL of pure water, and after dissolution, the pH value of the solution was adjusted to 7.0-7.2 with 1 mol / L NaOH solution. The medium was subjected to liquid sterilization in a high-pressure steam sterilization pot, and after sterilization was completed, it was divided into glass tubes, sealed and stored in a 4°C refrigerator for standby use.

[0092] The difference between the preparation method of the BL21(AI) E. coli containing ARG plasmid but not induced and surface modified with magnetic nanoparticles and Example 7 is that in the preparation of the BL21(AI) E. coli containing ARG plasmid but not induced and surface modified with magnetic nanoparticles, step (1) is that 1 mL of the glycerol bacteria carrying ARG plasmid prepared in Example 1 is inoculated into 200 mL of LB liquid medium 2, and cultured in a 37°C, 200 rpm shaker for 1.5 h, and then cultured in a 30°C, 160 rpm shaker for 22 h to obtain E. coli expressing nanobubble bag genes, and the remaining steps are described in Example 7.

[0093] 3 mL of three liquids were inoculated on the surface of the semi-solid medium, which were Figure 11 (a) BL21(AI) E. coli containing ARG plasmid but not induced (Example 1 prepared E. coli expressing nanobubble bag genes, the inoculated bacterial concentration was about 2×10 9 / mL), Figure 11 (b) only containing magnetic nanoparticles (the concentration of magnetic nanoparticles was about 10 mg / mL), and Figure 11 (c) BL21(AI) E. coli containing ARG plasmid but not induced and surface modified with magnetic nanoparticles (the inoculated bacterial concentration was about 2×10 9 / mL, and the concentration of magnetic nanoparticles was about 10 mg / mL), and the movement ability and anaerobic characteristics of each bacterial liquid were continuously observed.

[0094] Among them, the surface of the medium is in contact with air, which is the aerobic zone; the inside of the medium is isolated from air, which is the low-oxygen zone.

[0095] Figure 11 The results show that, Figure 11 the magnetic nanoparticles inoculated in (b) of Example 7 did not penetrate the semi-solid medium, Figure 11 (a) of Example 7 and Figure 11 (c) of Example 7 inoculated on the surface of the medium all penetrated the semi-solid medium and moved from the aerobic zone to the low-oxygen zone, indicating that the modification of magnetic nanoparticles on the surface of the bacteria can maintain the movement ability and anaerobic characteristics of the bacteria.

[0096] c、Animal experiment c-1, Construction of mouse tumor model: (1) The MCF-7 cells were digested using pancreatin, and after the cells were detached, an appropriate amount of PBS solution was added, and the cells were dispersed by gently blowing. The obtained MCF-7 cell suspension was centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. The cells were resuspended with PBS solution and washed twice with PBS solution to ensure the removal of residual pancreatin and other impurities. The supernatant was discarded, and the washed MCF-7 cells were obtained.

[0097] (2) The washed MCF-7 cells were resuspended in 1x PBS solution, and the cell concentration was adjusted to about 1x10 7 / mL to prepare a cell suspension.

[0098] (3) Construction of a mouse subcutaneous tumor model: 4-week-old Balb / c mice weighing about 16-20 g were selected, and 100 μL of the cell suspension prepared in step (2) was injected subcutaneously into the back of the thigh.

[0099] (4) Tumor growth monitoring: About two weeks after the injection of the cell suspension, when the tumor on the right leg of the mouse reached a diameter of about 10 mm, the mouse tumor model was successfully constructed, and subsequent experiments could be performed.

[0100] c-2, Mouse imaging First, the mouse was anesthetized, and then 100 μL of the bacterial complex suspension prepared in Example 2, with a concentration of 2x10 7 / mL, was injected into the tail vein of the tumor model mouse. After 12 h, the magnetic vibration imaging results at the tumor site of the mouse were observed by ultrasound imaging.

[0101] Figure 12 The gray-scale image of the image is the B-mode ultrasound imaging result, and the color part is the magnetic vibration imaging displacement result. The displacement of the tissue region without magnetic nanoparticles is 0, and the displacement of the tissue region with magnetic nanoparticles is not 0.

[0102] Figure 12 The results show that the bacterial complex modified with magnetic nanoparticles has a targeting effect on the tumor hypoxic area, and the nanobubble and magnetic nanoparticle carried by the bacterial complex can be detected to have gathered inside the tumor in the image.

[0103] d. Biological safety verification (1-1) Bacterial non-pathogenicity report The BL21 (AI) competent cells were purchased from Thermo Fisher, item number C607003. The company has passed the standard non-pathogenicity test to confirm its biological safety. The "Statement of nonpathogenicity" can be searched on the company's website.

[0104] (1-2) Bacterial plate growth experiment The tumor model mice prepared in step c-1 were injected with 100 μL of the bacterial complex suspension prepared in Example 2, which was surface-modified with magnetic nanoparticles, at a concentration of 2 x 10 7 The tumor model mice prepared in step c-1 were injected with 100 μL of the bacterial complex suspension prepared in Example 2, which was surface-modified with magnetic nanoparticles, at a concentration of 2 x 10

[0105] Figure 13 The results show that, compared with the control group, the acoustic-magnetic dual-mode diagnosis and treatment agent (test group) is distributed more in the tumor, and the E. coli in the tumor and liver tissue is rapidly reduced in the mouse body over time, and the tumor model mice of the test group survived after 1 day or 7 days of injection.

[0106] Example 9 The ultrasound energy was output through a single-element transducer with a diameter of 38 mm, and a circular cone-shaped ultrasound energy guide tube was placed at the front end of the transducer, which could guide the ultrasound energy into the cell culture dish. The bottom of the cell culture dish was A549 (human lung adenocarcinoma) cells cultured and the acoustic-magnetic dual-mode diagnosis and treatment agent prepared in Example 2 was added, and the number of A549 cells was 1:1 with the number of E. coli in the acoustic-magnetic dual-mode diagnosis and treatment agent prepared in Example 2. The effect of external ultrasound stimulation (center frequency: 1 MHz, negative peak acoustic pressure: 2.0 MPa, 5% duty cycle, cycle number: 5, pulse repetition frequency: 1 Hz) was detected by confocal microscopy (Nikon C2) for tumor cell membrane integrity before and after ultrasound stimulation, and acoustic response of the acoustic-magnetic dual-mode diagnosis and treatment agent.

[0107] Figure 14 The results show that, after external ultrasound excitation, the tumor cell membrane (Cellmask Orange orange fluorescent dye labeled) is no longer in the intact state of the (a) figure in Figure 14 , and the acoustic-magnetic dual-mode diagnosis and treatment agent (DMAO green fluorescent dye labeled) position in the (b) figure in Figure 14 appeared the perforation damage state of the (c) figure in Figure 14 , indicating that ultrasound stimulation caused tumor cell damage, thereby achieving the purpose of treating tumors.

[0108] Example 10 The tumor model mice prepared in step c-1 of Example 8 were injected with 100 μL of the bacterial complex suspension prepared in Example 2, which was surface-modified with magnetic nanoparticles, at a concentration of 2 x 10 7The temperature of the tumor region was detected by an infrared thermal imager (Fluke-Ti25) under the action of external magnetic field stimulation (field frequency: 2 MHz, electric power: 2 kW) for 10 minutes after injection of the bacterial complex suspension modified with magnetic nanoparticles prepared in Example 2 at a concentration of 1×109 / mL for 24 hours.

[0109] Figure 15 The results show that the acoustic-magnetic dual-mode diagnosis and treatment agent prepared by the present application is aggregated and released in the tumor region of the mouse (indicated by a black dotted line), the local magnetic heat effect induced by the magnetic particles, and the temperature of the tumor region exceeds 42℃, realizing magnetic heat treatment.

[0110] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A dual-mode sonomagnetic diagnostic and therapeutic agent, characterized in that, The acousto-magnetic dual-mode diagnosis and treatment agent is a surface modified magnetic nanoparticle of E. coli containing a nanobubble gene after induced expression.

2. The acoustomagnetic dual-mode theranostic agent of claim 1, wherein, The E. coli is E. coli BL21. The construction method of the E. coli containing the nanobubble gene comprises the following steps: The expression vector containing the nanobubble gene is transformed into the E. coli to obtain the E. coli containing the nanobubble gene.

3. The acoustomagnetic dual-mode theranostic agent of claim 2, wherein, The backbone vector of the expression vector is pBAD33.

4. The acoustomagnetic dual-mode theranostic agent of claim 1, wherein, The magnetic nanoparticle comprises a carboxyl magnetic nanoparticle or a streptavidin modified magnetic nanoparticle.

5. The acoustomagnetic dual-mode theranostic agent of claim 1, wherein, The surface modified magnetic nanoparticle of the E. coli containing the nanobubble gene after induced expression comprises any one of the following modes: (1) the carboxyl magnetic nanoparticle is modified on the surface of the E. coli containing the nanobubble gene after induced expression by an amide coupling method; (2) the biotinylated Sulfo-NHS is modified on the surface of the E. coli containing the nanobubble gene after induced expression, and the streptavidin modified magnetic nanoparticle is connected; (3) the biotinylated antibody is modified on the surface of the E. coli containing the nanobubble gene after induced expression, and the streptavidin modified magnetic nanoparticle is connected.

6. A method of preparing a dual-mode sonomagnetic diagnostic and therapeutic agent, characterized by, Any one of the following a-c is prepared: a, the carboxyl magnetic nanoparticle, EDC and Sulfo-NHS are mixed to obtain the activated magnetic nanoparticle; then the E. coli containing the nanobubble gene in any one of claims 1-5 after induced expression is incubated with the activated magnetic nanoparticle to obtain the surface modified magnetic nanoparticle of the E. coli containing the nanobubble gene after induced expression; b, the E. coli containing the nanobubble gene in any one of claims 1-5 after induced expression is incubated with the biotinylated Sulfo-NHS to obtain the surface biotinylated bacteria a, and then the surface biotinylated bacteria a is incubated with the streptavidin modified magnetic nanoparticle to obtain the surface modified magnetic nanoparticle of the E. coli containing the nanobubble gene after induced expression; c, the E. coli containing the nanobubble gene in any one of claims 1-5 after induced expression is incubated with the biotinylated antibody to obtain the surface biotinylated bacteria b, and then the surface biotinylated bacteria b is incubated with the streptavidin modified magnetic nanoparticle to obtain the surface modified magnetic nanoparticle of the E. coli containing the nanobubble gene after induced expression.

7. The preparation method according to claim 6, characterized in that, The induced expression comprises L-arabinose induced expression.

8. The preparation method according to claim 7, characterized in that, The concentration of the L-arabinose is 0.3-0.7 g / mL; the volume of the L-arabinose is 1-3 mL; and the induced expression is performed by placing in a 28-32℃, 150-170 rpm shaker for 20-24 hours.

9. The preparation method according to claim 6, characterized in that, In a, the incubation reaction is performed at 100-140 rpm for 15-25 min; In b, the first incubation reaction is performed at 100-140 rpm for 0.5-1.5 h, and the second incubation reaction is performed at 100-140 rpm for 0.5-1.5 h; In c, the first reaction is performed by incubation at 100-140 rpm for 0.5-1.5 h, and the second reaction is performed by incubation at 100-140 rpm for 0.5-1.5 h. The biotinylated Sulfo-NHS is EZ-link. TM Sulfo-NHS-LC-Biotin; the biotinylated antibody is a biotin-modified Escherichia coli serum type O / K polyclonal antibody.

10. Use of the acousto-magnetic dual-mode diagnosis and treatment agent of any one of claims 1-5 or the acousto-magnetic dual-mode diagnosis and treatment agent prepared by the preparation method of any one of claims 6-9 in the preparation of a tumor diagnosis and / or treatment agent.

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