Ultrasonic contrast agent for fallopian tubes of uterus and use thereof

Abstract

Provided in the present application are an ultrasonic contrast agent for fallopian tubes of a uterus and the use thereof. The contrast agent comprises the following components: a biosynthesized nanobubble, a nanobubble stabilizer and a physiological solvent. The nanobubble stabilizer is any one of a hyaluronic acid or a sodium salt thereof, an alginic acid or a sodium salt thereof, chitosan or gelatin. The nanobubble stabilizer in the contrast agent has a concentration of 0.025-0.2%. The OD500 of the contrast agent is 1-3.5. The contrast agent of the present application has the advantages of a low synthesis cost, a high mechanical index, a good imaging performance, etc., and can be used for ultrasonic contrast examination of diseases related to the fallopian tubes of the uterus.

Description

A uterine and fallopian tube ultrasound contrast agent and its application Technical Field

[0001] The present application belongs to the field of biomedicine, and specifically relates to a uterine and fallopian tube ultrasound contrast agent and its application. 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] Currently, there are two main types of hysterosalpingography used clinically: 1) X-ray angiography: A contrast agent is injected into the uterine cavity and fallopian tubes via a catheter, and X-ray fluoroscopy and radiography are performed. The contrast agent's visualization in the uterus, fallopian tubes, and pelvic cavity reveals the patency of the fallopian tubes, the location of obstruction, and the morphology of the uterine cavity. This procedure has the advantages of good visualization and minimal irritation, but it also has the disadvantages of long examination time, slow absorption, and a high risk of foreign body reactions. Furthermore, X-rays emit radiation, so even if the fallopian tubes are unobstructed after angiography, residual radiation from the contrast agent necessitates a wait of at least three months before considering pregnancy. 2) Ultrasound angiography: An ultrasound contrast agent is injected into the uterine cavity under transvaginal ultrasound guidance. The contrast agent's flow in the uterine cavity and fallopian tubes, as well as its diffusion after entering the pelvic cavity, is observed to determine any abnormalities in the uterine cavity, the patency of the fallopian tubes, and the normal course of the fallopian tubes. The contrast agent's flow and distribution can be observed in real time, revealing the patency of the fallopian tubes, the different morphologies of the fallopian tubes, and the presence of pelvic adhesions. Furthermore, CEUS offers clear images, is non-invasive, non-radioactive, has few adverse reactions, is brief, rarely causes allergic reactions, and is simple to perform. Assuming the fallopian tubes are unobstructed, pregnancy can be planned after one menstrual period has resumed. It is worth noting that the bolus pressure during hysterosalpingography can have a therapeutic effect on unclogging mild adhesions of the fallopian tubes, making it an advanced technology with both diagnostic and therapeutic benefits. In comparison, CEUS does not require X-rays and is simpler to perform, but the cost is generally higher.

[0004] At present, the uterine and fallopian tube ultrasound contrast agents commonly used in clinical practice in my country are mainly 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 2.5μm. Each bottle of contrast agent contains 25mg of lyophilized powder filled with sulfur hexafluoride gas. Before use, 5mL 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 50mg / mL. Each bottle contains 0.8×10 microspheres. 9 -2.2×10 9The average particle size is 3.0-4.5 μm. Microbubble contrast agents have a thin and soft phospholipid or protein shell membrane. 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 low 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 (MI, such as the SonoVue imaging MI of 0.08) of the acoustic beam, which is not conducive to scanning various sections for a long time.

[0005] Although other micro-nano ultrasound contrast agents have been developed in recent years, such as polymer microbubbles, acoustic liposomes, and mesoporous silica nanoparticles, these agents all have some drawbacks: for example, the membrane materials are complex and biocompatibility is poor. Furthermore, the preparation of these ultrasound contrast agents all uses chemical synthesis, which is complex and requires many steps. The resulting micro-nanobubbles have an unsatisfactory particle size distribution, and some contain excessive gas-encapsulating shell membranes, which prevent the contrast agents from releasing gas promptly within blood vessels or cavities, affecting the effectiveness of ultrasound contrast imaging. Therefore, there is an urgent need to develop more economical, effective, safe, and easy-to-prepare ultrasound contrast agents. Summary of the Invention

[0006] The present application provides a uterine and fallopian tube ultrasound contrast agent and its application. The contrast agent is prepared using biogas vesicles (also known as bionanobubbles) synthesized from natural and genetically engineered bubble-producing microorganisms. Unlike the chemically synthesized microbubbles currently used in clinical practice, it has the advantages of low synthesis cost, high mechanical index, and excellent imaging effect. It can be used for ultrasound contrast imaging of uterine and fallopian tube-related diseases. The contrast agent in this application has excellent in vitro and in vivo imaging effects, and the ultrasound contrast signal is uniform and strong.

[0007] In a first aspect, the present application provides a uterine and fallopian tube ultrasound contrast agent, the contrast agent comprising the following components: biosynthesized nanobubbles, a nanobubble stabilizer, and a physiological solvent;

[0008] The nanobubble stabilizer is any one of hyaluronic acid or its sodium salt, alginate or its sodium salt, chitosan or gelatin;

[0009] The concentration of the nanobubble stabilizer in the contrast agent is 0.025-0.2%, for example, 0.025%, 0.05%, 0.1%, 0.15% or 0.2%;

[0010] 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.

[0011] In the present application, the nanobubble stabilizer can also be any other injectable hydrogel, and the enhancement effect of different hydrogels is different. In the present application, hyaluronic acid or its sodium salt, alginic acid or its sodium salt, etc. have particularly good enhancement effects.

[0012] In this application, biosynthesized nanobubbles and nanobubble stabilizers are dissolved together in a physiological solvent for ultrasound contrast imaging, which can increase the in vitro and in vivo imaging effect of the contrast agent. Without the addition of a nanobubble stabilizer, the nanobubbles will initially be evenly dispersed in the physiological solvent, but some gas vesicles will float above the liquid under the action of buoyancy, affecting the imaging effect. Adding a nanobubble stabilizer can help the nanobubbles produce uniform contrast images on the one hand, and on the other hand, it can also increase the ultrasound contrast signal intensity of the nanobubbles.

[0013] Preferably, the physiological solvent is a physiologically tolerable aqueous dispersion medium.

[0014] Preferably, the physiological solvent is selected from physiological saline or PBS buffer.

[0015] In the present application, the physiological solvent can be selected from common injection solvents, such as physiological saline, phosphate buffer, glucose water, etc.

[0016] Preferably, the hyaluronic acid includes any one of macromolecular, medium molecular, small molecular or ultra-low molecular hyaluronic acid.

[0017] In this application, 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, it presents a colloidal state in the solution. Because of the presence of the colloid, the problem of uneven imaging 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.

[0018] In this application, the biological nanobubbles dispersed in a hyaluronic acid solution can produce a stronger ultrasound contrast signal with better signal uniformity. The contrast signal appears as a circular pore with a uniform internal signal. When the hyaluronic acid concentration in the contrast agent is 0.05%-0.2%, the contrast effect is good.

[0019] 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.

[0020] The amount of nanobubbles used in this application affects the imaging effect. The higher the concentration, the better. If the concentration is too low, the signal will be weak. If the concentration is too high, only the front will be developed, and the back will not be developed due to the attenuation of the sound. 500 When the OD value is within the range of 1-3.5, better imaging effect can be obtained. 500When the value is lower than 1.0, the ultrasound contrast imaging signal of nanobubbles is not obvious. 500 When the value is higher than 3.5, the nanobubble ultrasound contrast imaging signal produces an obvious crescent shape, and the posterior signal is not visualized.

[0021] 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%.

[0022] In this application, when the concentration of the nanobubble stabilizer in the contrast agent is less than 0.05%, the degree of enhancement 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 signal uniformity is poor. The preferred concentration range of the nanobubble stabilizer is 0.06-0.15%.

[0023] Preferably, the biosynthesized nanovesicles are selected from: air vesicle nanovesicles derived from natural vesicle-producing microorganisms and / or synthesized by genetically engineered vesicle-producing microorganisms.

[0024] Preferably, the biosynthetic nanobubbles are cylindrical or rugby-shaped, with a width of 45-250 nm and a length of 100-500 nm.

[0025] Second aspect: The present application provides a method for preparing the uterine and fallopian tube ultrasound contrast agent according to the first aspect, the preparation method comprising:

[0026] The biosynthesized nanobubbles are dispersed in a physiological solvent containing a nanobubble stabilizer to obtain a uterine and fallopian tube ultrasound contrast agent.

[0027] In the present application, 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 for subsequent imaging observation.

[0028] In a third aspect, the present application provides a 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.

[0029] The numerical range described in this application includes not only the point values ​​listed above, but also any point values ​​between the above numerical ranges that are not listed. Due to limited space and for the sake of simplicity, this application no longer exhaustively lists the specific point values ​​included in the range.

[0030] Compared with the prior art, this application has the following beneficial effects:

[0031] 1) The biosynthetic nanobubbles are used for preparation, which have better biocompatibility and are more environmentally friendly.

[0032] 2) Using hyaluronic acid as a stabilizer can enhance the contrast signal of nanobubbles and make the signal more uniform.

[0033] 3) It can tolerate a higher ultrasound mechanical index and have a longer imaging time. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is an electron micrograph of biosynthesized nanovesicles.

[0035] Figure 2 shows the in vitro ultrasound contrast imaging effects of biological nanobubbles of different concentrations and sources.

[0036] FIG3 is a graph showing the tolerable mechanical index test results of biological nanobubbles from different sources.

[0037] FIG4 shows the prepared uterine and fallopian tube ultrasound contrast agent.

[0038] FIG5 shows the in vitro imaging effect of contrast agents with different dosages of biological nanobubbles.

[0039] Figure 6 Imaging effects of ultrasound contrast agents in physiological saline or hyaluronic acid solutions of different concentrations.

[0040] FIG7 shows the imaging stability test results of ultrasound contrast agents.

[0041] FIG8 shows the ultrasound contrast imaging effect of the contrast agent of the present application on the rat uterus.

[0042] Figure 9 shows the ultrasound contrast imaging effect of biological nanobubbles in the rat fallopian tube.

[0043] Figure 10 In vitro effect experiment of uterine fallopian tube ultrasound contrast agent with different nanobubble stabilizers. DETAILED DESCRIPTION

[0044] The technical solution of the present application is further described below through specific implementation methods. Those skilled in the art should understand that the embodiments are only used to help understand the present application and should not be regarded as specific limitations of the present application.

[0045] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0046] Example 1

[0047] 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.

[0048] 1. Isolate and extract biological nanovesicles from natural vesicle-producing microorganisms (Halobacterium NRC 1).

[0049] (1) Add the vesicle-producing microorganisms to the culture medium and culture in a shaking incubator at 37°C (200 rpm, for 5-10 days).

[0050] (2) Place the culture medium in a separatory funnel and let it stand for 14-30 days until the bubbling microorganisms float on the surface of the liquid. Remove the lower layer of culture medium and separate the bubbling microorganisms.

[0051] (3) Add 2 times the volume of TMC lysis buffer to the isolated bubble-containing microorganisms for lysis overnight.

[0052] (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.

[0053] 2. Isolate and extract biological nanovesicles from genetically engineered bacteria (genetically engineered Escherichia coli carrying a vesicle-producing plasmid).

[0054] The synthetic gene cluster is derived from the Serratia air vesicle synthetic gene cluster, which includes gvpA1, gvpC, gvpN, gvpV, gvpF1, gvpG, gvpW, gvpA2, gvpK, gvpX, gvpA3, gvpY, gvrA, gvpH, gvpZ, gvpF2, gvpF3, gvrB and gvrC genes in sequence.

[0055] (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.

[0056] (2) The vesicle-producing genetically engineered bacteria were inoculated into liquid culture medium at a ratio of 1:100 and cultured in a shaking incubator at 37°C at 220 rpm until the OD 600 =0.6-0.8, add 0.4 M IPTG at a ratio of 1:1000; induce culture for 22-24 h.

[0057] (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). Centrifuge at 4°C, 500 g for 2 h, discard the supernatant, and wash with PBS. Repeat the same conditions three times to isolate the biological nanovesicles (biogas vesicles, GVs) and store them in a refrigerator at 4°C for later use.

[0058] Figure 1 is an electron micrograph of biosynthesized nanobubbles, (a) is a nanobubble synthesized from halophageal archaea, (b) is a nanobubble synthesized from genetically engineered bacteria, scale bar = 500 nm.

[0059] The results showed that both natural blister-producing halophilic archaea and genetically engineered blister-producing bacteria can synthesize biological gas vesicles (bio-nanovesicles) with a particle size of 100-300nm. The bio-nanovesicles synthesized by halophilic archaea are elliptical, while the bio-nanovesicles synthesized by genetically engineered blister-producing bacteria are long rod-shaped.

[0060] Example 2

[0061] This example investigates the ultrasound contrast imaging effects of biological nanobubbles from different sources.

[0062] In order to test whether biological nanobubbles can be used for ultrasound contrast imaging, in this example, the biosynthesized nanobubbles were diluted to different concentrations (Table 1) and in vitro imaging was performed using an ultrasound imager.

[0063] Table 1

[0064] Figure 2 shows the in vitro ultrasound contrast imaging effects of bio-nanovessels of different concentrations and sources. It can be seen that both bio-nanovessels derived from natural halophageal archaea (hGVs) and synthesized from genetically engineered bacteria (eGVs) have strong ultrasound contrast imaging effects, and the signal intensity increases with increasing concentration.

[0065] Example 3

[0066] This example investigates the mechanical index that biological nanobubbles from different sources can tolerate.

[0067] In order to test 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 imaging was performed under different mechanical index conditions (MI from 0.03-0.41) using an ultrasound imager.

[0068] Figure 3 shows the tolerable mechanical index test results of biological nanobubbles from different sources. The results show that the mechanical index of biological nanobubbles is significantly higher than that of commercial Sonovue microbubbles (when MI>0.1, Sonovue microbubbles will burst and the signal disappears). The tolerable mechanical index of biological nanobubbles from halophageal 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 biological nanobubbles synthesized from natural halophageal archaea (hGVs) and genetically engineered bacteria (eGVs) have higher ultrasonic mechanical index tolerance than commercial Sonovue.

[0069] Example 4

[0070] This embodiment provides a hysterosalpingography contrast agent, which comprises the following components: biosynthetic nanobubbles, hyaluronic acid (McLean, molecular weight 40-80 kD, 9004-61-9) and normal saline. The concentration of hyaluronic acid in the contrast agent is 0.1%; the OD of the contrast agent is 0.1%. 500 is 3.0.

[0071] The contrast agent is prepared as follows: biological nanobubbles are dispersed in physiological saline to an OD500 of 3.0, hyaluronic acid is added, and the mixture is uniformly mixed to obtain a hysterosalpingography ultrasound contrast agent. The mass percentage of hyaluronic acid in the contrast agent is 0.1%. The contrast agent is shown in FIG4 . The prepared hysterosalpingography ultrasound contrast agent is a milky white suspension.

[0072] Example 5

[0073] This embodiment provides a hysterosalpingography contrast agent, which comprises the following components: biosynthetic 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 0.1%. 500 is 3.0.

[0074] The preparation method of the contrast agent comprises the following steps: dispersing biological nanobubbles in a PBS buffer solution, adding hyaluronic acid, and mixing the mixture evenly to obtain a uterine and fallopian tube ultrasound contrast agent.

[0075] Example 6

[0076] This example investigates the effect of the amount of biological nanobubbles in the uterine and fallopian tube ultrasound contrast agent on the in vitro imaging effect.

[0077] The biological nanobubbles were dispersed in physiological saline, and hyaluronic acid (McLean, molecular weight 40-80 kD, 9004-61-9) was added to prepare the uterine fallopian tube ultrasound contrast agent according to Table 2.

[0078] Table 2

[0079] The contrast agent was placed in a 3% agar well and observed using an ultrasound imager in contrast mode. The results are shown in FIG5 .

[0080] Figure 5 shows the in vitro imaging effect of contrast agents with different dosages of biological nanobubbles. 500 When the value is lower than 1.0, the ultrasound contrast imaging signal of nanobubbles is not obvious. 500 When the concentration is higher than 3.5, the nanobubble ultrasound contrast imaging 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 nanobubbles used has a great influence on its in vitro imaging effect. 500 Only when it is within the range of 1-3.5 can better imaging effects be obtained.

[0081] The dosage of biological nanobubbles in this application 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.

[0082] Example 7

[0083] This example investigates the effect of the concentration of hyaluronic acid in the uterine and fallopian tube ultrasound contrast agent on in vitro imaging.

[0084] 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 in physiological saline and different hyaluronic acid concentrations (hyaluronic acid concentrations ranging from 0.025% to 0.2%).

[0085] OD 500 =3.0 biological nanobubbles are dispersed in normal saline, and a series of hyaluronic acid concentrations are added according to Table 3. After mixing evenly, the uterine and fallopian tube ultrasound contrast agent is obtained.

[0086] Table 3

[0087] The contrast agent was placed in a 3% agar well and observed using an ultrasound imager in contrast mode. The results are shown in FIG6 .

[0088] Figure 6 shows the imaging effect of ultrasound contrast agents in saline or hyaluronic acid solutions of varying concentrations. The results show that bio-nanobubbles dispersed in hyaluronic acid solutions produce stronger ultrasound contrast signals with improved signal uniformity. The contrast signals appear as circular pores with uniform internal signals. When the hyaluronic acid concentration in the contrast agent is between 0.05% and 0.2%, the contrast effect is excellent. When the hyaluronic acid concentration in the contrast agent is below 0.05%, the bio-nanobubble signal enhancement is limited. When the hyaluronic acid concentration in the contrast agent is above 0.2%, the bio-nanobubble signal is enhanced, but the signal uniformity is poor.

[0089] Adding nanobubble stabilizers can help nanobubbles produce contrast-enhanced images with uniform signals on the one hand, and on the other hand, it can also increase the ultrasound contrast signal intensity of nanobubbles.

[0090] The role of the hyaluronic acid or its sodium salt in the system is to enhance the imaging signal of the biological nanobubbles. At the same time, due to the presence of the colloid, the problem of uneven imaging signals caused by the floating of the nanobubbles in the physiological saline can be improved.

[0091] Example 8

[0092] This example investigates the imaging signal stability of the uterine and fallopian tube ultrasound contrast agent.

[0093] In order to further test the imaging stability of biological nanobubbles at different times, this example dispersed 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.

[0094] Figure 7 shows the imaging stability test results of the ultrasound contrast agent. In vitro phantom pore imaging results show that biological nanobubbles dispersed in a hyaluronic acid solution can maintain good contrast imaging stability, manifested by a more uniform imaging signal and slower signal decay (signal decay refers to the weakening of the signal due to the floating of nanobubbles over time). In comparison, the signal of biological nanobubbles dispersed in saline is relatively weaker. Over time, the signal uniformity gradually deteriorates, the outer circular outline is less obvious, and there is a black non-enhanced area inside.

[0095] Example 9

[0096] This example investigates the in vivo imaging effect of the uterine and fallopian tube ultrasound contrast agent.

[0097] 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 physiological saline were injected into the uterine cavity through the cervix of rats to perform uterine ultrasound imaging.

[0098] Figure 8 shows the contrast agent of this application in the uterus of rats undergoing ultrasound contrast imaging. Uterine ultrasound imaging was performed by injecting a mixture of biological nanobubbles with saline or varying concentrations of hyaluronic acid into the rat uterus. Clear ultrasound contrast imaging signals were observed within the uterus. Comparison revealed that the ultrasound contrast imaging signal generated by the mixture of biological nanobubbles and hyaluronic acid was more uniform and stronger.

[0099] A mixture of 0.1% hyaluronic acid and biological nanobubbles was injected into the fallopian tubes of rats through the uterine cavity for fallopian tube ultrasound imaging. It was also confirmed that obvious ultrasound contrast imaging signals could be observed in the rat fallopian tubes, as shown in Figure 9. Figure 9 shows the ultrasound contrast imaging effect of biological nanobubbles in the rat fallopian tubes.

[0100] Example 10

[0101] This embodiment provides a uterine fallopian tube ultrasound 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 0.1%. 500 is 3.0.

[0102] Example 11

[0103] This embodiment provides a hysterosalpingography contrast agent, which comprises 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 normal saline. The concentration of PVA in the contrast agent is 0.1%; the OD of the contrast agent is 0.1%. 500 is 3.0.

[0104] Example 12

[0105] This embodiment provides a uterine fallopian tube ultrasound contrast agent, which includes the following components: biosynthetic nanobubbles, chitosan (Aladdin, Lot#K1614016, CAS: 1398-61-4) and normal saline. The concentration of chitosan in the contrast agent is 0.1%; the OD of the contrast agent is 0.1%. 500 is 3.0.

[0106] The in vitro experimental results of the uterine and fallopian tube ultrasound contrast agents in Examples 10-12 are shown in FIG10 .

[0107] From the experimental results, it can be seen that by comparing the results of Example 7 with those of Example 10-12, the addition of hyaluronic acid, sodium alginate (in Example 10) or PVA (in Example 11) contrast agents can produce stronger ultrasound contrast signals, and the uniformity of the signal is also better, while the effect of the contrast agent of chitin (in Example 12) is not as good as the former, showing a weaker enhancement and uneven signal. The above results show that the nanobubble stabilizer in the present application can keep the nanobubble stable in the contrast agent for a long time, 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 in this application are all in line with safety.

[0108] In summary, the present application provides a biological nanobubble ultrasound contrast agent for uterine and fallopian tube use. The biological nanobubbles in the contrast agent are obtained by a biosynthesis method, have good biocompatibility, a simple synthesis process, low cost, and are nanoscale and uniform in 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.

[0109] The applicant declares that the above is only a specific implementation method of the present application, but the protection scope of the present application is not limited thereto. Technical personnel in the relevant technical field should understand that any changes or substitutions that can be easily thought of by technical personnel in the relevant technical field within the technical scope disclosed in this application fall within the protection scope and disclosure scope of this application.

Claims

1. A hysterosalpingographic contrast agent comprising the following components: biosynthetic nanobubbles, a nanobubble stabilizer, and a physiological solvent; in, The nanobubble stabilizer is any one of hyaluronic acid or its sodium salt, alginic acid 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 after dispersion in physiological solvent 500 1-3.

5.

2. The hysterosalpingography contrast agent according to claim 1, wherein: The physiological solvent is a physiologically tolerable aqueous dispersion medium.

3. The hysterosalpingography contrast agent according to claim 1, wherein: The physiological solvent is selected from physiological saline or PBS buffer.

4. The hysterosalpingography contrast agent according to claim 1, wherein The hyaluronic acid includes any one of macromolecular, medium molecular, small molecular or ultra-low molecular hyaluronic acid.

5. The hysterosalpingography contrast agent according to claim 1, wherein OD of the contrast agent 500 It is 2.5-3.

5.

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

7. The hysterosalpingography contrast agent according to claim 1, wherein: The biosynthesized nanovesicles are selected from: air vesicle nanovesicles derived from natural vesicle-producing microorganisms and / or synthesized by genetically engineered vesicle-producing microorganisms.

8. The hysterosalpingography 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 hysterosalpingographic contrast agent according to any one of claims 1 to 8, comprising: The biosynthesized nanobubbles are dispersed in a physiological solvent and a nanobubble stabilizer is added to obtain a uterine and 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 a product for ultrasound contrast examination of uterine and fallopian tube related diseases.

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