Uterine fallopian tube ultrasonic contrast agent and application thereof

Abstract

The invention provides a uterine fallopian tube ultrasonic contrast agent and application thereof. The contrast agent is prepared from the following components: biosynthetic nano-bubbles, a nano-bubble stabilizer and a physiological solvent, the nano foam stabilizer is any one of hyaluronic acid or sodium salt thereof, alginic acid or sodium salt thereof, chitosan or gelatin; the concentration of the nano bubble stabilizer in the contrast agent is 0.025 to 0.2 percent; the OD500 of the contrast agent is 1 to 3.5. The contrast agent provided by the invention has the advantages of low synthesis cost, high mechanical index, good imaging effect and the like, and can be used for ultrasonic contrast examination of uterine fallopian tube related diseases.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to a uterine fallopian tube ultrasonic contrast agent and application thereof. Background Art

[0002] Uterine lesions and fallopian tube obstruction are one of the main causes of female infertility. Hysterosalpingography is an effective method to check the patency of female fallopian tubes and has been widely used in clinical practice.

[0003] At present, there are two main types of hysterosalpingography in clinical practice: 1) X-ray contrast examination: contrast agent is injected into the uterine cavity and fallopian tube through a catheter, and X-ray fluoroscopy and filming are performed. According to the contrast agent's appearance in the uterine fallopian tube and pelvic cavity, the patency of the fallopian tube, the location of obstruction and the morphology of the uterine cavity are understood. It has good development effect and little stimulation, but there are problems such as long inspection time, slow absorption and easy to cause foreign body reaction. Moreover, X-ray has radiation. Even if the fallopian tube is unobstructed after contrast, it is necessary to wait at least three months before considering pregnancy due to the residual radiation of contrast agent. 2) Ultrasound contrast examination: Ultrasound contrast agent is injected into the uterine cavity under the guidance of transvaginal ultrasound. By observing the flow of contrast agent in the uterine cavity and fallopian tube and the diffusion after entering the pelvic cavity, it is judged whether there are abnormalities in the uterine cavity, whether the fallopian tube is unobstructed, and whether the fallopian tube runs normally. The flow and distribution of contrast agent can be observed in real time to understand whether the fallopian tube is unobstructed, the different morphologies of fallopian tubes and pelvic adhesions. In addition, ultrasound angiography has clear images, is non-invasive, non-radioactive, has low adverse reactions, is short in duration, has few allergic reactions, and is easy to operate. If the fallopian tubes are unobstructed, pregnancy can be prepared after one menstruation is restored after ultrasound angiography. It is worth pointing out that the push pressure during hysterosalpingography can have a certain therapeutic effect on the dredging of slightly adhered fallopian tubes, and it is an advanced technology that has both diagnostic and therapeutic effects. In comparison, fallopian tube ultrasound angiography does not require exposure to X-rays and is easier to operate, but the cost is generally higher.

[0004] At present, the uterine fallopian tube ultrasound contrast agents commonly used in clinical practice in my country mainly include SonoVue produced by Bracco of Switzerland and Lidaxin produced by Xiamen Lipin Pharmaceutical Co., Ltd. SonoVue is a lipid microbubble with an average particle size of 2.5 microns. Each bottle of contrast agent contains 25 mg of lyophilized powder filled with sulfur hexafluoride gas. Before use, 5 ml of normal saline is added and shaken for 2 minutes to form a microbubble suspension. It is currently the most widely used product. Lidaxin is a perfluoropropane human albumin microsphere with an albumin content of 50 mg / mL. Each bottle contains 0.8×10 microspheres. 9 -2.2×10 9The average particle size is 3.0-4.5μm; microbubble contrast agents have thin and soft phospholipid or protein shell membranes. Under the action of low sound pressure, the microbubbles have good resonance characteristics and can generate strong harmonic signals, which can obtain real-time harmonic images with lower noise. However, this micron-sized microbubble contrast agent has high preparation costs and short imaging time. It can only exist in organs under the condition of a low mechanical index (mechanical index, MI, such as the SonoVue imaging mechanical index of 0.08), which is not conducive to scanning each section for a long time.

[0005] Although other ultrasound contrast agents such as polymer microbubbles, acoustic liposomes, mesoporous silicon nanoparticles, and micro-nano ultrasound contrast agents have been developed at home and abroad in recent years, these ultrasound contrast agents all have some problems: for example, the composition of the membrane material is complex and the biocompatibility is poor. In addition, the preparation process of these ultrasound contrast agents is synthesized by chemical methods, which is complicated and the steps are cumbersome. The particle size distribution of the prepared micro-nano bubbles is not ideal. Some of the shell membrane materials that encapsulate the gas have too many layers, which causes the contrast agent to be unable to release the gas in time in the blood vessels or cavities, affecting the effect of ultrasound contrast. Therefore, it is urgent to develop more economical, effective, safe and easy-to-prepare ultrasound contrast agents. Summary of the invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a uterine fallopian tube ultrasound contrast agent and its application. The contrast agent is prepared by using biological air sacs (also called biological nanobubbles) synthesized by natural bubble-producing microorganisms and genetically engineered bubble-producing microorganisms. It is different from the chemically synthesized microbubbles currently used in clinical practice. It has the advantages of low synthesis cost, high mechanical index, and good imaging effect, and can be used for ultrasound contrast examination of uterine fallopian tube related diseases. The contrast agent in the present invention has a good in vitro and in vivo imaging effect, and the ultrasound contrast signal is uniform and strong.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a uterine fallopian tube ultrasound contrast agent, the contrast agent comprising the following components: biosynthesized nanobubbles, a nanobubble stabilizer, and a physiological solvent;

[0009] The nanobubble stabilizer is any one of hyaluronic acid or its sodium salt, alginate or its sodium salt, chitosan or gelatin;

[0010] The concentration of the nanobubble stabilizer in the contrast agent is 0.025-0.2%, for example, it can be 0.025%, 0.05%, 0.1%, 0.15% or 0.2%;

[0011] The OD of the contrast agent 500 It is 1-3.5, for example, it can be 1, 2, 2.5, 3 or 3.5, etc.

[0012] In the present invention, the nanobubble stabilizer can also be any other injectable hydrogel, and different hydrogels have different effects on enhancing imaging. In the present invention, hyaluronic acid or its sodium salt, alginate or its sodium salt, etc. have particularly good enhancing effects.

[0013] In the present invention, the biosynthesized nanobubbles and the nanobubble stabilizer are dissolved together in a physiological solvent, and ultrasound contrast imaging is performed, which can increase the in vitro and in vivo imaging effect of the contrast agent. When the nanobubble stabilizer is not added, the nanobubbles will be uniformly dispersed in the physiological solvent at the beginning, but some gas vesicles will float above the liquid under the action of buoyancy, affecting the imaging effect. Adding the nanobubble stabilizer can help the nanobubbles produce a uniform contrast image on the one hand, and on the other hand, it can also improve the ultrasound contrast signal intensity of the nanobubbles.

[0014] Preferably, the physiological solvent is a physiologically tolerable aqueous dispersion medium.

[0015] Preferably, the physiological solvent is selected from physiological saline or PBS buffer.

[0016] In the present invention, the physiological solvent can be selected from common injection solvents, such as physiological saline, phosphate buffer, glucose water, etc.

[0017] Preferably, the hyaluronic acid includes any one of macromolecular, medium molecular, small molecular or ultra-low molecular hyaluronic acid.

[0018] In the present invention, the role of the hyaluronic acid or its sodium salt in the system is to enhance the imaging signal of biological nanobubbles, and at the same time present a colloidal state in the solution. Because of the presence of the colloid, the problem of uneven contrast signal caused by the floating of nanobubbles in physiological saline can be improved. Hyaluronic acid of various molecular weights can achieve the above signal enhancement effect.

[0019] In the present invention, biological nanobubbles dispersed in a hyaluronic acid solution can generate stronger ultrasound contrast signals, and the signal uniformity is also better. The contrast signal is in the shape of a circular gel hole, and the internal signal is uniform. When the concentration of hyaluronic acid in the contrast agent is 0.05%-0.2%, the contrast effect is good.

[0020] Preferably, the OD of the contrast agent is 500 It is 2.5-3.5, for example, it can be 2.5, 3 or 3.5, etc.

[0021] The amount of nanobubbles used in the present invention affects the imaging effect. The higher the concentration, the better. If the concentration is too low, the signal is weak. If the concentration is too high, only the front is developed, and the back is not developed due to the attenuation of the sound. 500 When the OD value is within the range of 1-3.5, a better imaging effect can be obtained. 500When it is lower than 1.0, the nanobubble ultrasound contrast signal is not obvious. 500 When it is higher than 3.5, the nanobubble ultrasound contrast signal produces an obvious crescent shape, and the posterior signal is not visualized.

[0022] Preferably, the concentration of the nanobubble stabilizer in the contrast agent is 0.06-0.15%, for example, 0.06%, 0.1%, 0.12%, 0.14% or 0.15%.

[0023] In the present invention, when the concentration of the nanobubble stabilizer in the contrast agent is lower than 0.05%, the enhancement degree of the biological nanobubble signal is limited. When the concentration of the nanobubble stabilizer in the contrast agent is higher than 0.2%, the biological nanobubble signal is enhanced, but the uniformity of the signal is poor. The preferred range of the concentration of the nanobubble stabilizer is 0.06-0.15%.

[0024] Preferably, the biosynthesized nanovesicles are selected from: gas vesicle nanovesicles derived from natural vesicle-producing microorganisms and / or synthesized by genetically engineered vesicle-producing microorganisms.

[0025] Preferably, the biosynthetic nanobubble is cylindrical or rugby-shaped, with a width of 45-250 nm and a length of 100-500 nm.

[0026] In a second aspect, the present invention provides a method for preparing the uterine fallopian tube ultrasound contrast agent according to the first aspect, the preparation method comprising:

[0027] The biosynthesized nanobubbles are dispersed in a physiological solvent containing a nanobubble stabilizer to obtain a uterine fallopian tube ultrasound contrast agent.

[0028] In the present invention, the preparation method of the contrast agent is simple and can be used immediately after preparation. The nanobubbles can be stably dispersed in the solution and will not float for a long time, which is beneficial to subsequent imaging observation.

[0029] In a third aspect, the present invention provides use of the uterine and fallopian tube ultrasound contrast agent described in the first aspect in the preparation of an ultrasound contrast examination product for uterine and fallopian tube related diseases.

[0030] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1) The biosynthesized nanobubbles are used for preparation, which have better biocompatibility and are more environmentally friendly.

[0033] 2) Using hyaluronic acid as a stabilizer can enhance the contrast signal of nanobubbles and make the signal more uniform.

[0034] 3) Able to tolerate a higher ultrasound mechanical index and longer contrast imaging time. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is an electron micrograph of biosynthesized nanovesicles.

[0036] Figure 2 The in vitro ultrasound contrast imaging effects of biological nanobubbles of different concentrations and sources.

[0037] Figure 3 This is a graph showing the tolerable mechanical index test results of biological nanobubbles from different sources.

[0038] Figure 4 The invention discloses a uterine and fallopian tube ultrasound contrast agent prepared.

[0039] Figure 5 The in vitro imaging effect of contrast agents with different dosages of biological nanobubbles.

[0040] Figure 6 Imaging effect of ultrasound contrast agent in normal saline or hyaluronic acid solution of different concentrations.

[0041] Figure 7 These are the imaging stability test results of ultrasound contrast agents.

[0042] Figure 8 The ultrasonic contrast imaging effect of the contrast agent of the present invention in the rat uterus.

[0043] Fig. 9 This is the ultrasound contrast imaging effect of biological nanobubbles in the rat fallopian tube.

[0044] Fig.10 In vitro effect experiment of hysterosalpingography contrast agent with different nanobubble stabilizers. DETAILED DESCRIPTION

[0045] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0046] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0047] Example 1

[0048] This example uses the natural vesicle-producing microorganism Halobacterium NRC 1 and genetically engineered Escherichia coli carrying a vesicle-producing plasmid to synthesize biological nanovesicles.

[0049] 1. Isolate and extract biological nanovesicles from natural vesicle-producing microorganisms (Halobacterium NRC 1).

[0050] (1) Add the vesicle-producing microorganisms to the culture medium and culture them in a shaking incubator at 37°C (200 rpm for 5-10 days).

[0051] (2) The culture medium is placed in a separatory funnel and allowed to stand for 14-30 days until the vesicular microorganisms float to the surface of the liquid. The lower layer of culture medium is removed to separate the vesicular microorganisms.

[0052] (3) Add 2 volumes of TMC lysis buffer to the separated bubble-containing microorganisms for lysis overnight.

[0053] (4) The lysed solution was centrifuged at 200-500 g for 4 h, the lower layer was removed by aspiration, and the centrifugation and washing were repeated three times under the same conditions to separate the bio-nanovesicles (gas vesicles, GVs) and store them in a refrigerator at 4°C for future use.

[0054] 2. Isolate and extract biological nanovesicles from genetically engineered bacteria (genetically engineered Escherichia coli carrying a vesicle-producing plasmid).

[0055] The synthetic gene cluster is derived from the Serratia air vesicle synthetic gene cluster, and includes gvpA1, gvpC, gvpN, gvpV, gvpF1, gvpG, gvpW, gvpA2, gvpK, gvpX, gvpA3, gvpY, gvrA, gvpH, gvpZ, gvpF2, gvpF3, gvrB and gvrC genes in sequence.

[0056] (1) The synthetic gene cluster of the biological nanovesicle is cloned into a prokaryotic expression plasmid vector, and then the recombinant plasmid containing the synthetic gene cluster of the biological nanovesicle is transferred into competent Escherichia coli cells to obtain a vesicle-producing genetically engineered bacterium.

[0057] (2) The vesicle-producing genetically engineered bacteria were inoculated into liquid culture medium at a ratio of 1:100 and cultured in a shaker at 37°C at 220 rpm until the OD 600 =0.6-0.8, add 0.4M IPTG at a ratio of 1:1000; induce culture for 22-24h.

[0058] (3) Collect the bacteria containing vesicles by centrifugation (4°C, 4000 rpm, 30 min) and resuspend them in SoluLyse-Tris bacterial lysis buffer (L200500 Genlantis) for lysis. Centrifuge at 4°C, 500 g for 2 h, discard the supernatant, and wash with PBS. Repeat the same conditions for 3 times to isolate the biological nanovesicles (biogas vesicles, GVs) and store them in a 4°C refrigerator for future use.

[0059] Figure 1 Electron micrographs of biosynthesized nanovesicles, (a) nanovesicles synthesized from halophilic archaea, (b) nanovesicles synthesized from genetically engineered bacteria, scale bar = 500 nm.

[0060] The results showed that both natural halophilic archaea and genetically engineered vesicle-producing bacteria can synthesize biological air vesicles (biogenic nanovesicles) with a particle size of 100-300nm. The biological nanovesicles synthesized by halophilic archaea are elliptical, while the biological nanovesicles synthesized by genetically engineered vesicle-producing bacteria are long rods.

[0061] Example 2

[0062] This example investigates the ultrasound contrast imaging effects of biological nanobubbles from different sources.

[0063] In order to test whether biological nanobubbles can be used for ultrasound contrast imaging, in this example, the biologically synthesized nanobubbles were diluted to different concentrations (Table 1) and an ultrasound imager was used for in vitro imaging.

[0064] Table 1

[0065]

[0066]

[0067] Figure 2 The in vitro ultrasound contrast imaging effects of biological nanobubbles of different concentrations and sources. It can be seen that the biological nanobubbles derived from natural halophage archaea (hGVs) and synthesized from genetically engineered bacteria (eGVs) have strong ultrasound contrast imaging effects, and the signal intensity increases with the increase of concentration.

[0068] Example 3

[0069] This example investigates the mechanical index that biological nanobubbles from different sources can tolerate.

[0070] In order to detect the mechanical index tolerance of biological nanobubbles, this example uses biological nanobubbles from two sources to prepare OD 500 =3.0 concentration, with commercial SonoVue (8 μL / mL) as control, placed in 3% agar wells, and ultrasound contrast imaging was performed using an ultrasound imager under different mechanical index conditions (MI ranging from 0.03-0.41).

[0071] Figure 3 The results of the tolerable mechanical index test of biological nanobubbles from different sources are shown in the figure. The results show that the mechanical index of biological nanobubbles imaging is significantly higher than that of commercial Sonovue microbubbles (when MI>0.1, Sonovue microbubbles will be exploded and the signal disappears). The tolerable mechanical index of biological nanobubbles from halophagous archaea is 0.22, while the tolerable mechanical index of biological nanobubbles from genetically engineered bubble-producing bacteria is 0.32. The best ultrasonic mechanical index of commercial Sonovue is 0.08. It can be seen that the biological nanobubbles synthesized from natural halophagous archaea (hGVs) and genetically engineered bacteria (eGVs) have higher ultrasonic mechanical index tolerance than the commercial Sonovue.

[0072] Example 4

[0073] This embodiment provides a hysterosalpingography contrast agent, which includes the following components: biosynthesized nanobubbles, hyaluronic acid (McLean, molecular weight 40-80 kD, 9004-61-9) and physiological saline. The concentration of hyaluronic acid in the contrast agent is 0.1%; the OD of the contrast agent is 500 is 3.0.

[0074] The preparation method of the contrast agent is as follows: disperse the biological nanobubbles in physiological saline, the OD500 of the dispersed solution is 3.0, add hyaluronic acid, mix evenly to obtain the uterine fallopian tube ultrasound contrast agent, the mass percentage of hyaluronic acid in the contrast agent is 0.1%. Figure 4 As shown, the prepared uterine fallopian tube ultrasound contrast agent is a milky white suspension.

[0075] Example 5

[0076] This embodiment provides a hysterosalpingography contrast agent, which includes the following components: biosynthesized nanobubbles, hyaluronic acid and PBS buffer. The concentration of hyaluronic acid (McLean, molecular weight 30-45 kD, 9004-61-9) in the contrast agent is 0.1%; the OD of the contrast agent is 500 is 3.0.

[0077] The preparation method of the contrast agent comprises: dispersing biological nanobubbles in PBS buffer, adding hyaluronic acid, and mixing evenly to obtain the uterine fallopian tube ultrasound contrast agent.

[0078] Example 6

[0079] This example investigates the effect of the amount of biological nanobubbles in the uterine fallopian tube ultrasound contrast agent on the in vitro imaging effect.

[0080] The biological nanobubbles were dispersed in physiological saline, hyaluronic acid (McLean, molecular weight 40-80 kD, 9004-61-9) was added, and the uterine fallopian tube ultrasound contrast agent was prepared according to Table 2.

[0081] Table 2

[0082]

[0083]

[0084] The above contrast agent was placed in a 3% agar well and observed using an ultrasound imager in contrast mode. Figure 5 shown.

[0085] Figure 5 The in vitro imaging effect of the contrast agent with different dosages of biological nanobubbles. 500 When it is lower than 1.0, the nanobubble ultrasound contrast signal is not obvious. 500 When the concentration is higher than 3.5, the nanobubble ultrasound contrast signal produces an obvious crescent shape, and the rear signal is not developed, indicating that the concentration of the nanobubble is too high. This shows that the amount of biological nanobubble used has a great influence on its in vitro imaging effect. 500 When it is within the range of 1-3.5, better imaging effect can be obtained.

[0086] The dosage of the biological nanobubbles in the present invention affects the imaging effect. The higher the concentration, the better. If the concentration is too low, the signal is weak. If the concentration is too high, only the front is imaged, and the back is not imaged (black shadow) due to the attenuation of sound.

[0087] Example 7

[0088] This example investigates the effect of the concentration of hyaluronic acid in the uterine and fallopian tube ultrasound contrast agent on the in vitro imaging effect.

[0089] Since ultrasound contrast agents easily float in aqueous solutions, resulting in uneven ultrasound contrast signals, this example further tests the imaging signal stability of biological nanobubbles under physiological saline and different hyaluronic acid concentrations (hyaluronic acid concentrations ranging from 0.025% to 0.2%).

[0090] OD 500 =3.0 biological nanobubbles are dispersed in physiological saline, and a series of concentrations of hyaluronic acid are added according to Table 3, and mixed evenly to obtain uterine fallopian tube ultrasound contrast agent.

[0091] Table 3

[0092]

[0093]

[0094] The above contrast agent was placed in a 3% agar well and observed using an ultrasound imager in contrast mode. Figure 6 shown.

[0095] Figure 6 The imaging effect of ultrasound contrast agent in physiological saline or hyaluronic acid solution of different concentrations. The results showed that biological nanobubbles dispersed in hyaluronic acid solution can produce stronger ultrasound contrast signals, and the signal uniformity is also better. The contrast signal appears in the shape of a circular pore, and the internal signal is uniform. When the concentration of hyaluronic acid in the contrast agent is 0.05%-0.2%, the contrast effect is good. When the concentration of hyaluronic acid in the contrast agent is lower than 0.05%, the degree of enhancement of the biological nanobubble signal is limited. When the concentration of hyaluronic acid in the contrast agent is higher than 0.2%, the biological nanobubble signal is enhanced, but the signal uniformity is poor.

[0096] Adding nanobubble stabilizers can help nanobubbles produce contrast images with uniform signals on the one hand, and on the other hand, it can also increase the ultrasound contrast signal intensity of nanobubbles.

[0097] The role of the hyaluronic acid or its sodium salt in the system is to enhance the imaging signal of biological nanobubbles. At the same time, due to the presence of colloids, the problem of uneven contrast signals caused by the floating of nanobubbles in physiological saline can be improved.

[0098] Example 8

[0099] This example investigates the imaging signal stability of the uterine fallopian tube ultrasound contrast agent.

[0100] In order to further test the imaging stability of biological nanobubbles at different times, the present embodiment dispersed the biological nanobubbles in physiological saline or hyaluronic acid solution (OD 500 =3.0), and in vitro imaging was performed at 0 sec, 1 min, and 5 min after the addition of the phantom wells.

[0101] Figure 7 The results of the imaging stability test of ultrasound contrast agents. The results of in vitro phantom pore imaging show that biological nanobubbles dispersed in hyaluronic acid solution can maintain good contrast imaging stability, which is manifested by more uniform imaging signals and slower signal decay (signal decay means that as time goes by, the nanobubbles will float and cause the signal to weaken). In comparison, the signal of biological nanobubbles dispersed in physiological saline is relatively weaker. As time goes by, the signal uniformity gradually deteriorates, the outer circular outline is not obvious, and there is a black non-enhanced area inside.

[0102] Example 9

[0103] This example investigates the in vivo imaging effect of the uterine fallopian tube ultrasound contrast agent.

[0104] 1. In order to further verify whether the ultrasound contrast agent can be used for uterine cavity imaging, in this example, biological nanobubbles dispersed in different concentrations of hyaluronic acid or saline were injected into the uterine cavity through the cervix of rats to perform uterine ultrasound imaging.

[0105] Figure 8 The contrast agent of the present invention is used for ultrasound contrast imaging in rat uterus. A mixture of biological nanobubbles and saline or hyaluronic acid of different concentrations is injected into the rat uterine cavity for uterine ultrasound imaging, and obvious ultrasound contrast imaging signals can be observed in the uterus. By comparison, it is found that the ultrasound contrast signal generated by the mixture of biological nanobubbles and hyaluronic acid is more uniform and stronger.

[0106] 2. A mixture of 0.1% hyaluronic acid and biological nanobubbles was injected into the fallopian tubes of rats for ultrasound imaging of the fallopian tubes. It was also confirmed that obvious ultrasound contrast imaging signals could be observed in the fallopian tubes of rats, such as Fig. 9 As shown, Fig. 9 This is the ultrasound contrast imaging effect of biological nanobubbles in the rat fallopian tube.

[0107] Example 10

[0108] This embodiment provides a hysterosalpingography contrast agent, which includes the following components: biosynthetic nanobubbles, sodium alginate (McLean, M:G=2:1, 9004-38-3) and normal saline. The concentration of sodium alginate in the contrast agent is 0.1%; the OD of the contrast agent is 500 is 3.0.

[0109] Embodiment 11

[0110] This embodiment provides a hysterosalpingography contrast agent, which includes the following components: biosynthetic nanobubbles, polyvinyl alcohol (PVA) (Sigma-Aldrich, Lot#SLBS6476, 87-90% hydrolyzed, high molecular weight average molecular weight 30,000-70,000) and physiological saline. The concentration of PVA in the contrast agent is 0.1%; the OD of the contrast agent is 500 is 3.0.

[0111] Example 12

[0112] This embodiment provides a hysterosalpingography contrast agent, which includes the following components: biosynthetic nanobubbles, chitosan (Aladdin, Lot#K1614016, CAS: 1398-61-4) and saline. The concentration of chitosan in the contrast agent is 0.1%; the OD of the contrast agent is 500 is 3.0.

[0113] The in vitro effect experimental results of the uterine fallopian tube ultrasound contrast agent in Examples 10-12 are as follows Fig.10 shown.

[0114] From the experimental results, it can be seen that by comparing the results of Example 7 with those of Examples 10-12, the contrast agent added with hyaluronic acid, sodium alginate (in Example 10) or PVA (in Example 11) can produce a stronger ultrasound contrast signal, and the uniformity of the signal is also better, while the contrast agent of chitin (in Example 12) is not as good as the former, showing weaker enhancement and uneven signal. The above results show that the nanobubble stabilizer in the present invention can keep the nanobubble stable for a long time in the contrast agent, can be evenly dispersed, and is not easy to float on the upper part of the liquid, and has good fluidity, can flow in the cavity, and realize hysterosalpingography, and the reagents selected by the present invention are all in line with safety.

[0115] In summary, the present invention provides a biological nanobubble ultrasound contrast agent for uterine and fallopian tubes. The biological nanobubbles in the contrast agent are obtained by a biosynthesis method, have good biocompatibility, simple synthesis process, low cost, nanoscale and uniform particle size; the prepared contrast agent has good imaging effect and good stability, and has important application prospects in the detection of uterine and fallopian tube lesions.

[0116] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A hysterosalpingographic contrast agent, characterized in that: The contrast agent comprises the following components: biosynthesized nanobubbles, a nanobubble stabilizer and a physiological solvent; The nanobubble stabilizer is any one of hyaluronic acid or its sodium salt, alginate or its sodium salt, polyvinyl alcohol or gelatin; The concentration of the nanobubble stabilizer in the contrast agent is 0.025-0.2%; The OD of the biosynthesized nanobubbles in the contrast agent dispersed in a physiological solvent 500 It is 1-3.

5.

2. The hysterosalpingographic contrast agent according to claim 1, characterized in that: The physiological solvent is a physiologically tolerable aqueous dispersion medium.

3. The hysterosalpingographic contrast agent according to claim 1, characterized in that: The physiological solvent is selected from physiological saline or PBS buffer.

4. The hysterosalpingographic contrast agent according to claim 1, characterized in that: The hyaluronic acid includes any one of macromolecular, medium molecular, small molecular or ultra-low molecular hyaluronic acid.

5. The hysterosalpingographic contrast agent according to claim 1, characterized in that: The OD of the contrast agent 500 It is 2.5-3.

5.

6. The hysterosalpingographic contrast agent according to claim 1, characterized in that: The concentration of the nanobubble stabilizer in the contrast agent is 0.06-0.15%.

7. The hysterosalpingographic contrast agent according to claim 1, characterized in that: The biosynthesized nanovesicles are selected from: gas vesicle nanovesicles derived from natural vesicle-producing microorganisms and / or synthesized gas vesicle nanovesicles from genetically engineered vesicle-producing microorganisms.

8. The hysterosalpingographic contrast agent according to claim 1, characterized in that: The biosynthesized nanobubbles are cylindrical or rugby-shaped, with a width of 45-250 nm and a length of 100-500 nm.

9. A method for preparing the uterine fallopian tube ultrasound contrast agent according to any one of claims 1 to 8, characterized in that: The preparation method comprises: The biosynthesized nanobubbles are dispersed in a physiological solvent, and a nanobubble stabilizer is added to obtain a uterine fallopian tube ultrasound contrast agent.

10. Use of the uterine and fallopian tube ultrasound contrast agent according to any one of claims 1 to 8 in the preparation of an ultrasound contrast examination product for uterine and fallopian tube related diseases.

Images