Gastrointestinal ultrasonic dual-display contrast agent and application thereof

Abstract

The invention provides a gastrointestinal ultrasonic dual-display contrast agent and application thereof. The contrast agent is a mixed solution of a biological source gas vesicle solution and edible cereal powder particles, the volume ratio of the gas vesicle solution in the mixed solution is 0.2-0.3%, and the OD500 of the gas vesicle solution is 1.5-3.5; the concentration of the edible cereal powder particles in the mixed solution is 0.01-0.1 g / mL; the gas vesicles are derived from microorganisms containing air sacs; the airbag-containing microorganisms comprise halophilic archaea, escherichia coli, listeria monocytogenes or algae; the edible cereal powder particles are prepared from edible plant powder and an adhesive. The contrast agent is simple in preparation method, not easy to coagulate, uniform, stable and good in imaging effect; better stability and acid resistance are achieved, and it can be ensured that the contrast agent keeps stable in a gastral cavity and provides a lasting imaging signal.

Description

Technical Field

[0001] The invention belongs to the field of medicine and diagnosis technology, and in particular relates to a gastrointestinal ultrasound dual-display contrast agent and application thereof. Background Art

[0002] Gastrointestinal diseases have brought a heavy burden to people's lives and health, and prevention and treatment are imminent. From a histological analysis, most gastrointestinal diseases are accompanied by obvious changes in the morphology of the gastrointestinal wall, which can be used as a basis for disease diagnosis. For example, gastric ulcers can erode the stomach wall, causing changes in the morphology of the stomach wall. Using this principle, gastroscopy and barium meal contrast are currently commonly used in clinical practice to examine gastric diseases, but patients may experience adverse reactions such as regurgitation and gastric spasm. Some examinations also have electromagnetic radiation, and the acceptance of the examination is low. In recent years, gastrointestinal filling ultrasound examination has emerged for gastrointestinal diseases. Ultrasound contrast imaging is non-invasive and radiation-free, low-cost, convenient and fast. It is currently the most popular medical imaging technology and can be widely used in the diagnosis of gastrointestinal diseases.

[0003] Gastrointestinal ultrasound contrast examination is also known as gastrointestinal filling examination. It is an ultrasound examination method that uses oral or enema contrast agents to fill the gastrointestinal tract and eliminate the interference of gas and contents in the cavity with ultrasound, thereby improving the effect of gastrointestinal ultrasound imaging and clearly showing the hierarchical structure of the gastrointestinal wall and lesions. The contrast agents used in gastrointestinal ultrasound contrast examination can be divided into two types: echoless and echogenic. Echogenic contrast agents stay in the gastrointestinal tract for a longer time and can significantly reduce the interference of artifacts caused by gas in the gastrointestinal tract. Currently, there are authorized invention patents for oral echogenic contrast agents that use rice, wheat, sesame and other grains as the main raw materials, or add microbubble ultrasound contrast agent suspensions to enhance ultrasound imaging effects. Lipid-based microbubbles are a widely used ultrasound contrast agent. Their particle size is usually 1-5μm. The large particle size provides a strong contrast signal. However, due to the relatively fragile physical and chemical properties of phospholipid microbubbles, the preparation process of this type of contrast agent is relatively complicated, the cost is high, and it is difficult to adapt to the acidic environment of the stomach.

[0004] There are problems with the gastrointestinal ultrasound contrast agents currently used in clinical practice: ① The preparation method is relatively cumbersome, requiring boiling water and rapid stirring, which is easy to coagulate and uneven, affecting the imaging effect of the contrast agent; ② The preparation time is relatively long, which increases the waiting time for patients. ③ It is easy to stratify in a short period of time and cannot form a uniform paste. It is easy to form light spots and cause artifacts, thus affecting the diagnostic effect. ③ The physical and chemical properties of phospholipid microbubbles are relatively fragile, which makes the preparation process of this type of contrast agent more complicated, high cost, and difficult to adapt to the acidic environment of the stomach.

[0005] Therefore, developing a gastrointestinal contrast agent that is easy to prepare and low-cost is of great significance for improving the diagnosis and treatment efficiency of clinical gastrointestinal diseases. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a gastrointestinal ultrasound dual-display contrast agent and its application. The present invention combines an echogenic contrast agent with gas vesicles from bacteria to prepare a novel oral gastrointestinal contrast agent that synergistically enhances ultrasound contrast signals.

[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 gastrointestinal ultrasound dual-display contrast agent, wherein the contrast agent is a mixed solution of a biologically derived gas vesicle solution and edible grain powder particles;

[0009] The volume ratio of the gas vesicle solution in the mixed solution is 0.2-0.3%, for example, 0.2%, 0.25% or 0.3%, etc. The OD of the gas vesicle solution is 500 1.5-3.5, for example, 1.5, 2, 2.5, 3 or 3.5, etc.;

[0010] The concentration of the edible grain powder particles in the mixed solution is 0.01-0.1 g / mL, for example, 0.01 g / mL, 0.02 g / mL, 0.04 g / mL, 0.05 g / mL, 0.06 g / mL, 0.08 g / mL or 1 g / mL, etc.;

[0011] The gas vesicles are derived from gas-vesicle-containing microorganisms; the gas-vesicle-containing microorganisms include halophilic archaea, Escherichia coli, Listeria or algae;

[0012] The edible grain powder particles are prepared from edible plant powder and a binder.

[0013] The present invention has a synergistic effect of echogenic gastrointestinal contrast agent and nano-scale ultrasound contrast agent GVs (gas vesicles), which can make GVs stably suspended in the solution, with bright echoes and stronger signals. Compared with ordinary echogenic gastrointestinal contrast agents, the present invention can simultaneously form B-ultrasound and contrast signals, which provides richer diagnostic information for clinical practice.

[0014] Preferably, in the raw materials for preparing the edible grain powder particles, the weight proportion of edible plant powder is 60-90 parts, for example, it can be 60 parts, 65 parts, 70 parts, 75 parts, 80 parts, 85 parts or 90 parts, etc., and the weight proportion of adhesive is 10-40 parts, for example, it can be 10 parts, 15 parts, 20 parts, 25 parts, 30 parts, 35 parts or 40 parts, etc.

[0015] In the present invention, the ratio between the edible plant powder and the adhesive effectively improves the stability of the preparation, and the prepared preparation is not easy to be stratified, thereby achieving more uniform imaging in the gastrointestinal tract.

[0016] Preferably, the particle size of the gas vesicles is 100-600 nm, for example, it can be 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm or 600 nm.

[0017] In the present invention, nanoscale GVs generally have better stability and acid resistance than microbubbles. Digestive juices such as gastric acid may affect contrast agents. Stability and acid resistance can ensure that the contrast agents remain stable in the gastric cavity and provide persistent imaging signals.

[0018] Preferably, the gas vesicles are solidified gas vesicles.

[0019] In order to further improve the mechanical index of the biological gas vesicles, the present invention also solidifies the gas vesicles. The mechanical index (MI) is an indicator of the intensity of ultrasound. It is an indicator of the tolerance of gas vesicles to ultrasound. A higher MI value means that the intensity of ultrasound is greater, which may cause the gas vesicles to rupture or degrade, thereby affecting the imaging quality and contrast.

[0020] Preferably, the curing is glutaraldehyde curing, and the steps of the glutaraldehyde curing include:

[0021] Glutaraldehyde is mixed with the gas vesicle solution, and then allowed to stand for the first time after the mixing; a glycine solution is added for neutralization, and then allowed to stand for the second time; and unreacted glutaraldehyde, glycine and reaction byproducts thereof are removed.

[0022] In the present invention, the glutaraldehyde-cured gas vesicles have higher mechanical stability in clinical applications than uncured vesicles. The curing process enhances the acoustic pressure tolerance of the structure by cross-linking the shell of the vesicles, enabling them to maintain integrity at higher ultrasound intensities, thereby providing stable imaging contrast under high MI conditions.

[0023] Preferably, the final concentration of glutaraldehyde in the mixed solution is 0.5-2%, such as 0.5%, 1%, 1.5%, 2% or 2.5%, etc.; preferably 0.5-1.5%, such as 1%, 1.2%, 1.3%, 1.4% or 1.5%, etc.

[0024] In the present invention, during the process of adding glutaraldehyde and mixing, uniform distribution of glutaraldehyde should be ensured to promote uniform cross-linking of the vesicle wall.

[0025] Preferably, the first standing time is 12-36 hours, for example, 12, 16, 18, 24, 30 or 36 hours.

[0026] In the present invention, the curing time after adding glutaraldehyde is preferably 12-36 hours, and sufficient standing time can ensure the effect of cross-linking and curing.

[0027] In the present invention, the glycine is used to neutralize glutaraldehyde.

[0028] Preferably, the neutralization time is 6-12 hours, for example, 6, 8, 10 or 12 hours.

[0029] Preferably, the unreacted glutaraldehyde, glycine and reaction by-products thereof are removed by dialysis.

[0030] In a specific embodiment, the dialysis treatment time is not less than 24 hours; the dialysis fluid of the dialysis treatment is 0.9% normal saline; the molecular weight cutoff (or pore size) of the dialysis treatment filter membrane is 14KD.

[0031] Preferably, the edible plant powder is selected from any one of corn powder, coix seed powder, soybean powder, wheat powder or sesame powder, or a combination of at least two of them.

[0032] Preferably, the adhesive is selected from any one of lotus root starch, pectin, linseed gum, carboxymethyl cellulose or locust bean gum, or a combination of at least two thereof.

[0033] In the present invention, the sieve particle size of the edible plant powder is 80-100 mesh, for example, it can be 80, 90 or 100 mesh.

[0034] In the present invention, the sieve particle size of the adhesive is 80-100 mesh, for example, 80, 90 or 100 mesh.

[0035] In the present invention, the granular solid particles are uniform, the solution is not easy to stratify, and the stability is high. During ultrasound imaging, the stable suspension allows the high-echo particles to be evenly distributed and not condensed into agglomerates. The taste is delicate and the subject's compliance is high; the brewing process is simple and convenient, and it can be brewed with drinking water of different temperatures, which reduces the waiting time of the subject and improves the efficiency of clinical examination.

[0036] In a preferred embodiment of the present invention, the edible plant powder is a combination of corn powder and coix seed powder; and the adhesive is lotus root powder.

[0037] In the present invention, the sieved particle size of the edible cereal powder particles is 0.5-1.5 mm, for example, it can be 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm or 1.5 mm.

[0038] In the present invention, the particles obtained by screening with a 0.5-1.5 mm sieve dissolve quickly and evenly without being easily agglomerated.

[0039] In the present invention, the edible grain powder particles are prepared by a granulation process. The granulation process is a wet granulation process. The wet granulation process includes: mixing edible plant powder and a binder in proportion, adding a wetting agent to form a soft material; passing the soft material through a sieve to form wet granules, and then passing the sieve to obtain a granular solid after drying.

[0040] In the present invention, the size of the sieve is 0.5-1.5 mm, for example, it can be 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm or 1.5 mm, etc.

[0041] In the present invention, the drying temperature is 60-80°C, for example, 60°C, 65°C, 70°C, 75°C or 80°C.

[0042] In the present invention, the wetting agent is selected from: ethanol solution, starch slurry or cellulose derivatives.

[0043] In the present invention, the mass fraction of the ethanol solution is 50-75%, for example, it can be 50%, 55%, 60%, 65%, 70% or 75%.

[0044] In the present invention, the gas vesicles can generate strong contrast signals through ultrasound, which can effectively improve the contrast of ultrasound imaging. The gas vesicles synthesized by the commonly used chassis cells in molecular biology such as Escherichia coli and Listeria modified by genetic engineering technology can be replaced.

[0045] In the present invention, the gastrointestinal ultrasound contrast agent is easy to use. The prepared granular solid is mixed with water to form a solution, and then the solution is mixed evenly with the gas vesicle solution before use. In different use cases, the preparation amount can be proportionally expanded according to needs. The gastrointestinal ultrasound contrast agent has a delicate taste and high compliance of the examinee; the brewing process is simple and convenient, and it can be brewed with drinking water of different temperatures, which reduces the waiting time of the examinee and improves the efficiency of clinical examination.

[0046] The gas vesicles described in the present invention are prepared by a method comprising the following steps: culturing halophilic archaea to separate microorganisms containing gas vesicles from a culture solution, lysing the microorganisms, and collecting the released gas vesicles by centrifugation.

[0047] In the present invention, the culture conditions are: culture at 37±2°C in a shaker at a speed of 200-220 rpm for 5-8 days.

[0048] In the present invention, the lysis solution used for the lysis includes, by concentration, 8-12 mM Tris-HCl, 2-3 mM MgCl2 and 1.5-2.5 mM CaCl2, pH 7.0-7.5.

[0049] In the present invention, the centrifugal conditions are: 0-4°C, 250-350g centrifugation for 2.5-4 hours.

[0050] In a second aspect, the present invention provides a method for preparing the gastrointestinal ultrasound dual-display contrast agent described in the first aspect, the preparation method comprising: mixing edible grain powder particles and biologically derived gas vesicles with water to obtain the gastrointestinal ultrasound dual-display contrast agent.

[0051] In the present invention, the OD of the gas vesicles before and after dilution is 500 The value control is as follows: 10 mL of gas vesicle solution was added to 500 mL of water to obtain the OD 500 The OD of the gas vesicle solution before dilution is taken as an example. 500 The values ​​are 1.5, 2.0, 2.5, 3.0, 3.5; the OD of the diluted solution 500 The values ​​are 0.058, 0.065, 0.091, 0.157, and 0.397.

[0052] In a preferred embodiment of the present invention, 10 mL OD 500 The gas vesicle solution with a value of 3.0 is added to 500 mL of water, and then 30 g of edible grain powder particles are added and mixed to obtain a gastrointestinal ultrasound dual-display contrast agent.

[0053] In the present invention, the OD of the gas vesicles in the gastrointestinal ultrasound dual-display contrast agent is 500 In the range of 0.05-0.5, the image is clear and the contrast is high. 500 Outside the range, it is not conducive to showing the complete gastrointestinal tract boundaries.

[0054] The OD of the gas vesicles described in the present invention is 500 0.05-0.5 refers to the OD value measured after adding the gas vesicle solution to water when preparing the gastrointestinal ultrasound dual-display contrast agent. 500 0.05-0.5, which is the OD before adding edible grain powder particles 500 .

[0055] In a third aspect, the present invention provides use of the gastrointestinal ultrasound dual-display contrast agent described in the first aspect in the preparation of an ultrasound gastrointestinal contrast examination product.

[0056] Preferably, the ultrasound gastrointestinal angiography examination includes any one of or a combination of at least two of gastrointestinal motility function detection, gastric ulcer detection, gastric space-occupying detection, ulcerative colitis detection or Crohn's disease detection.

[0057] The present invention provides a novel gastrointestinal contrast agent, which can be used for ultrasonic gastrointestinal contrast examination, including but not limited to: gastrointestinal motility function, gastric ulcer, gastric mass, ulcerative colitis, Crohn's disease, etc.

[0058] 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.

[0059] Compared with the prior art, the present invention has the following beneficial effects:

[0060] (1) Nanoscale GVs generally have better stability and acid resistance than microbubbles. Digestive fluids such as gastric acid may affect contrast agents. Stability and acid resistance can ensure that contrast agents remain stable in the gastric cavity and provide long-lasting imaging signals.

[0061] (2) The echogenic gastrointestinal contrast agent and the nanoscale ultrasound contrast agent GVs act synergistically. The echogenic gastrointestinal ultrasound contrast agent can enhance the contrast imaging signal intensity of GVs and can stably suspend GVs in the solution with bright echoes. Compared with ordinary echogenic gastrointestinal contrast agents, the present invention can simultaneously form B-ultrasound and contrast signals, which provides richer diagnostic information for clinical practice.

[0062] (3) The particles are uniform, the solution is not easy to stratify, and the stability is high. During ultrasound imaging, the stable suspension obtained after preparation allows the high-echo particles to be evenly distributed, not coagulated into clumps, not easy to empty in the stomach, and the imaging time is long.

[0063] (4) The taste is delicate and the subjects are highly compliant; the brewing process is simple and convenient and can be brewed with drinking water at different temperatures, which reduces the waiting time of the subjects and improves the efficiency of clinical examinations.

[0064] (5) The gas vesicles in the contrast agent of the present invention are solidified gas vesicles, which can withstand higher mechanical pressure. In the detection, the signal intensity begins to gradually decrease when the MI reaches 0.6. This indicates that the solidification treatment enhances the mechanical stability of the gas vesicles, so that it can still effectively enhance the image quality and contrast under high ultrasound intensity imaging conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1This is an ultrasound imaging picture of the rat stomach after the new gastrointestinal contrast agent was gavaged into the rat.

[0066] Figure 2 This is a graph showing the internal sedimentation results of the gastrointestinal ultrasound dual-display contrast agent prepared from different edible grain powder particles.

[0067] Figure 3 This is the super imaging result of gastrointestinal ultrasound dual-display contrast agent prepared from different edible grain powder particles.

[0068] Figure 4 This is a graph showing the in-tube sedimentation results of gastrointestinal ultrasound dual-display contrast agents prepared with different adhesives.

[0069] Figure 5 The following are the super imaging results of gastrointestinal ultrasound dual-display contrast agents prepared with different adhesives.

[0070] Figure 6 These are the B-ultrasound imaging results of contrast agents prepared with different proportions of components.

[0071] Figure 7 The B-ultrasound imaging results of contrast agents prepared with different gas vesicle concentrations.

[0072] Figure 8 The figures show the B-ultrasound and contrast imaging results of contrast agents prepared from vesicles from different sources.

[0073] Fig. 9 The effect of different mechanical indices on the contrast signal of uncured and cured gas vesicles.

[0074] Fig.10 This is a graph showing the results of investigating the conditions for solidifying gas vesicles with glutaraldehyde. DETAILED DESCRIPTION

[0075] 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.

[0076] 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.

[0077] Example 1

[0078] This embodiment provides a gastrointestinal ultrasound dual-display contrast agent, which is a mixed solution of a biological gas vesicle solution and edible grain powder particles. The volume ratio of the gas vesicle solution in the mixed solution is 0.2%. The OD of the gas vesicle solution is500 is 3.0 (the final OD of the gas vesicles in the mixed solution 500 is 0.16); the concentration of the edible grain powder particles is 0.06g / mL, and the solvent is water; the particle size of the gas vesicles is 200-300nm, and the gas vesicles are derived from halophilic archaea (Halobacteria sp.NRC-1).

[0079] 1. The preparation method of the gastrointestinal ultrasound dual-display contrast agent comprises: mixing a biological gas vesicle solution, edible grain powder particles and water to obtain the gastrointestinal ultrasound dual-display contrast agent.

[0080] (A) The method for preparing the gas vesicle solution comprises:

[0081] (1) Add Halobacteria sp. NRC-1 (Halo) to ATCC culture medium and culture it in a shaker at 37°C at 220 rpm for 5-8 days. Then place the culture medium in a separatory funnel and let it stand until the microorganisms float to the surface. Remove the lower culture medium and separate the microorganisms containing GVs.

[0082] (2) Add an equal volume of TMC lysis buffer (10 mM Tris-HCl, 2.5 mM MgCl2 and 2 mM CaCl2, pH 7.5) to the isolated microorganisms. Divide the lysed microorganisms into centrifuge tubes and centrifuge at 300 g for 4 h at 4°C.

[0083] (3) The unlysed microorganisms were aspirated with a syringe, PBS was added again, and centrifugation was repeated until all the microorganisms were completely lysed. The released GVs were transferred to a new centrifuge tube and stored at 4°C for future use.

[0084] (B) Preparation of edible grain flour particles

[0085] (1) Grind the cooked product through a 100-mesh sieve.

[0086] (2) The three ingredients are mixed in a mass ratio of 40:40:20, and 75% ethanol is added to prepare a soft material that can be kneaded into a ball and dispersed when pressed.

[0087] (3) Screening by extrusion to form wet granules.

[0088] (4) Spread evenly in an oven or drying oven and dry at 70°C.

[0089] (5) After drying, sieve through a 0.8 mm sieve to obtain particles with uniform particle size.

[0090] 2. How to use oral gastrointestinal ultrasound contrast agents

[0091] The gas vesicle solution, the granular solid and water are mixed for in vivo ultrasound imaging; 500 =3 gas vesicle solution 10 mL and 30 g of edible grain powder particles and 500 mL of water were mixed to obtain a gastrointestinal ultrasound dual-display contrast agent.

[0092] When used orally, slowly pour 30g of the prepared granular solid into 500mL of drinking water. The temperature of the drinking water can be adjusted according to personal preference. Stir evenly while adding. Add 10mL (OD 500 =3), mix well and it can be used. The gastrointestinal capacity of the subjects varies, so the dosage can be increased or decreased as appropriate.

[0093] When used for enema, pour 60g of granular solid into 1200mL of water and stir evenly; add 20mL (OD 500 =3), mix well and it can be used.

[0094] 3. Extracorporeal ultrasound imaging

[0095] The new gastrointestinal contrast agent was placed in the 1% agarose model hole, and the linear array probe (3-11MHz) of the clinical ultrasound diagnostic equipment Mindray Reson 7 was placed on one side of the agarose model. The imaging was performed in the ultrasound contrast imaging mode, which can generate strong and stable contrast ultrasound signals. It can be seen that the ultrasound contrast signals of only echo-type gastrointestinal contrast agents and only GVs are extremely weak, while the signal of the new gastrointestinal contrast agent mixed with the two is significantly enhanced, and as the concentration of GVs increases, its ultrasound signal becomes stronger.

[0096] 4. In vivo ultrasound imaging

[0097] The new gastrointestinal ultrasound contrast agent solution was injected into the stomach of male SD rats for in vivo ultrasound imaging, which showed uniform imaging and clear gastric cavity edges. Figure 1 )

[0098] Example 2

[0099] This embodiment provides an oral gastrointestinal ultrasound contrast agent. The preparation method of the contrast agent is different from that of embodiment 1 in that the ratio of corn flour, coix seed powder and lotus root powder is 37:37:26; the volume ratio of the gas vesicle solution in the mixed solution is 0.3%; the OD of the gas vesicle solution is 500 The concentration of edible grain powder particles in the mixed solution is 0.1 g / mL. The preparation method of gas vesicles and granular solids is as described in Example 1.

[0100] Example 3

[0101] This embodiment provides a gastrointestinal ultrasound dual-display contrast agent. The only difference between this embodiment and Example 1 is that the edible grain powder particles are prepared from rice powder and lotus root powder, and the remaining steps refer to Example 1.

[0102] Example 4

[0103] This embodiment provides a gastrointestinal ultrasound dual-display contrast agent. The only difference between this embodiment and Example 1 is that the edible grain powder particles are prepared from coix seed powder and lotus root powder, and the remaining steps refer to Example 1.

[0104] Example 5

[0105] This embodiment provides a gastrointestinal ultrasound dual-display contrast agent. The only difference between this embodiment and Example 1 is that the edible grain powder particles are prepared from corn powder and lotus root powder, and the remaining steps refer to Example 1.

[0106] Example 6

[0107] This embodiment provides a gastrointestinal ultrasound dual-display contrast agent. The only difference between this embodiment and Example 1 is that the edible grain powder particles are prepared from yam powder and lotus root powder, and the remaining steps refer to Example 1.

[0108] Example 7

[0109] This embodiment provides a gastrointestinal ultrasound dual-display contrast agent. The only difference between this embodiment and Example 1 is that the edible grain powder particles are prepared from soybean powder and lotus root powder, and the remaining steps refer to Example 1.

[0110] In the above embodiment, the gastrointestinal ultrasound dual-display contrast agent prepared with rice flour, coix seed flour, corn flour, yam flour and soybean flour was investigated for in vitro sedimentation and B-ultrasound imaging effects, wherein the effects were investigated when the ratio of powder to water was 4%, 6% and 8%, respectively.

[0111] Figure 2 The results of internal sedimentation of gastrointestinal ultrasound dual-display contrast agents prepared from different edible grain powder particles are shown in the figure. From the figure, we can see that the grain powder is mainly used to investigate whether the imaging effect is uniform, and the sedimentation effect is mainly determined by the glue.

[0112] Figure 3 The results of super imaging of gastrointestinal ultrasound dual-display contrast agents prepared from different edible grain powder particles. As can be seen from the figure, in the imaging analysis, the distribution of rice powder shows heterogeneity, and the significant high signal points in its image are unevenly distributed. The imaging signals of yam powder and soybean powder are relatively weak, and the imaging uniformity is poor. In contrast, coix seed powder and corn powder show higher imaging signal intensity, and the imaging results show good uniformity and fineness, making the imaging quality of these two powders significantly better than the former.

[0113] Example 8

[0114] This embodiment provides a gastrointestinal ultrasound dual-display contrast agent. The only difference between this embodiment and Example 1 is that the edible grain powder particles are prepared from corn powder, coix seed powder, and locust bean gum. The remaining steps refer to Example 1.

[0115] Example 9

[0116] This embodiment provides a gastrointestinal ultrasound dual-display contrast agent. The only difference between this embodiment and Example 1 is that the edible grain powder particles are prepared from corn powder, coix seed powder and guar gum, and the remaining steps refer to Example 1.

[0117] Example 10

[0118] This embodiment provides a gastrointestinal ultrasound dual-display contrast agent. The only difference between this embodiment and Example 1 is that the edible grain powder particles are prepared from corn powder, coix seed powder and konjac gum, and the remaining steps refer to Example 1.

[0119] Embodiment 11

[0120] This embodiment provides a gastrointestinal ultrasound dual-display contrast agent. The only difference between this embodiment and Example 1 is that the edible grain powder particles are prepared from corn powder, coix seed powder, and Prussian gum. The remaining steps refer to Example 1.

[0121] The gastrointestinal ultrasound dual-display contrast agents prepared with different adhesives in the above embodiments were investigated for their in vitro sedimentation and B-ultrasound imaging effects.

[0122] Figure 4 The results of the in-tube sedimentation of gastrointestinal ultrasound dual-display contrast agents prepared with different adhesives are shown in the figure. From left to right, the viscosity of the five kinds of gums mixed with powders, including locust bean gum, guar gum, konjac gum, pullulan gum and lotus root powder, is different. As can be seen from the figure, all test groups settled within 5 minutes, among which the sedimentation rate of the guar gum group was close to 30%, and the sedimentation rates of the other groups except lotus root powder were all around 20%; the sedimentation rate of the lotus root powder group was only about 4%, indicating that the contrast agent in the lotus root powder group has the characteristics of uniform suspension and high viscosity, which can improve the imaging quality and prolong the gastrointestinal emptying time during the examination.

[0123] Figure 5The results of ultrasound imaging of gastrointestinal ultrasound dual-display contrast agents prepared with different adhesives are shown in the figure. From left to right, the ultrasound imaging of five kinds of gums and powders, including locust bean gum, guar gum, konjac gum, pullulan gum, and lotus root powder, before and after sedimentation. It can be observed from the image that locust bean gum, guar gum, konjac gum, and pullulan gum present many high-echo signal points in ultrasound imaging. The distribution of these signal points is uneven, resulting in rough imaging quality and lack of fineness. In addition, during the static imaging process, these colloids also showed signal stratification. In contrast, almost no high-echo signal points were detected in the lotus root powder group, and no sedimentation phenomenon was observed in the imaging effect.

[0124] Example 12

[0125] This embodiment provides a gastrointestinal ultrasound dual-display contrast agent. The only difference between this embodiment and Example 1 is that the ratio of the edible plant powder to the adhesive is 60:40, wherein the edible plant powder is a mixture of corn flour and coix seed powder in equal mass; the remaining steps refer to Example 1.

[0126] Example 13

[0127] This embodiment provides a gastrointestinal ultrasound dual-display contrast agent. The only difference between this embodiment and Example 1 is that the ratio of the edible plant powder to the adhesive is 70:30, wherein the edible plant powder is a mixture of corn flour and coix seed powder in equal mass; the remaining steps refer to Example 1.

[0128] Embodiment 14

[0129] This embodiment provides a gastrointestinal ultrasound dual-display contrast agent. The only difference between this embodiment and Example 1 is that the ratio of the edible plant powder to the adhesive is 80:20, wherein the edible plant powder is a mixture of corn flour and coix seed powder in equal mass; the remaining steps refer to Example 1.

[0130] Embodiment 15

[0131] This embodiment provides a gastrointestinal ultrasound dual-display contrast agent. The only difference between this embodiment and Example 1 is that the ratio of the edible plant powder to the adhesive is 90:10, wherein the edible plant powder is a mixture of corn flour and coix seed powder in equal mass; the remaining steps refer to Example 1.

[0132] The gastrointestinal ultrasound dual-display contrast agents prepared from edible plant powder and adhesive in different proportions in the above embodiment were subjected to B-ultrasound imaging effects. The specific experimental results are as follows: Figure 6 shown.

[0133] Figure 6The B-ultrasound imaging results of contrast agents prepared with different proportions of components. It can be observed from the image that when the mixing ratio of plant powder and adhesive is 80:20, the prepared contrast agent shows uniform and delicate image quality in B-ultrasound imaging and has a high signal intensity. In contrast, contrast agents with other proportions have high echo signal points during the imaging process, and these signal points are unevenly distributed, resulting in a low overall signal intensity. Therefore, the ratio of 80:20 may be the optimal ratio to achieve the best imaging effect.

[0134] Comparative Example 1

[0135] This embodiment provides a gastrointestinal ultrasound dual-display contrast agent. The only difference between this embodiment and embodiment 1 is that the contrast agent comprises, in terms of concentration: 0.06 g / mL edible grain powder particles, the solvent is water, and no gas vesicles are contained therein.

[0136] Example 16

[0137] This embodiment provides a gastrointestinal ultrasound dual-display contrast agent. The difference between this embodiment and embodiment 1 is that the volume ratio of the gas vesicle solution in the mixed solution is 0.2%. The OD of the gas vesicle solution is 500 is 1.5 (the final OD of the gas vesicles in the mixed solution 500 is 0.05), and the concentration of edible grain powder particles is 0.06g / mL.

[0138] Embodiment 17

[0139] This embodiment provides a gastrointestinal ultrasound dual-display contrast agent. The difference between this embodiment and embodiment 1 is that the volume ratio of the gas vesicle solution in the mixed solution is 0.2%. The OD of the gas vesicle solution is 500 =2.0 (the final OD of gas vesicles in the mixed solution 500 is 0.06), and the concentration of edible grain powder particles is 0.06g / mL.

[0140] Embodiment 18

[0141] This embodiment provides a gastrointestinal ultrasound dual-display contrast agent. The difference between this embodiment and embodiment 1 is that the volume ratio of the gas vesicle solution in the mixed solution is 0.2%. The OD of the gas vesicle solution is 500 is 2.5 (the final OD of the gas vesicles in the mixed solution 500 is 0.09), and the concentration of edible grain powder particles is 0.06g / mL.

[0142] Embodiment 19

[0143] This embodiment provides a gastrointestinal ultrasound dual-display contrast agent. The difference between this embodiment and embodiment 1 is that the volume ratio of the gas vesicle solution in the mixed solution is 0.2%. The OD of the gas vesicle solution is 500 =3 (the final OD of gas vesicles in the mixed solution 500 is 0.16), and the concentration of edible grain powder particles is 0.06g / mL.

[0144] Embodiment 20

[0145] This embodiment provides a gastrointestinal ultrasound dual-display contrast agent. The difference between this embodiment and embodiment 1 is that the volume ratio of the gas vesicle solution in the mixed solution is 0.2%. The OD of the gas vesicle solution is 500 3.5 (the final OD of gas vesicles in the mixed solution 500 is 0.4), and the concentration of edible grain powder particles is 0.06g / mL.

[0146] In the above embodiment, the gastrointestinal ultrasound dual-display contrast agent with different gas vesicle concentrations was used to perform B-ultrasound and contrast imaging. The specific experimental results are as follows: Figure 7 shown.

[0147] Figure 7 The following is a diagram of the B-ultrasound imaging results of contrast agents prepared with different gas vesicle concentrations. According to the graphical data, it can be observed that when the optical density (OD) value reaches 0.16 or higher, the signal intensity of the contrast agent is significantly improved, and the imaging becomes more uniform. In addition, the OD value does not have a significant effect on the B-ultrasound imaging signal. Therefore, under the premise of ensuring that the contrast agent has a high signal intensity and uniformity, selecting a contrast agent with an OD value above 0.16 will obtain a better imaging effect, that is, the final OD of the mixed solution obtained by dissolving the gas vesicle solution in water is 500 The contrast agent has better imaging effect when it is greater than 0.16.

[0148] Embodiment 21

[0149] This example investigates the effects of different sources or types of vesicles in gastrointestinal ultrasound dual-display contrast agents on the contrast agent effect. The preparation steps of the gastrointestinal ultrasound dual-display contrast agent refer to Example 1, and the sources of the vesicles used include: Halo (abbreviation of Halobacteria sp. NRC-1), BL21 (E. coli), MG1655 (E. coli), and Nissle 1917 (E. coli).

[0150] The above-mentioned gastrointestinal ultrasound dual-display contrast agent was used to investigate the effects of B-ultrasound and contrast imaging. The specific experimental results are as follows: Figure 8 shown.

[0151] Figure 8 The contrast agents prepared from vesicles of different sources show the B-ultrasound and contrast imaging results of the contrast agents. When the vesicles are derived from Halo, the obtained contrast agent shows the strongest signal intensity in contrast imaging and the best imaging uniformity. In contrast, contrast agents prepared from vesicles of other sources show almost the same performance in B-ultrasound signals. Therefore, in application scenarios that pursue high imaging quality and signal uniformity, choosing Halo-derived vesicles as the preparation material of contrast agents will be able to obtain better imaging effects.

[0152] Embodiment 22

[0153] This embodiment provides a gastrointestinal ultrasound dual-display contrast agent. The difference between this embodiment and embodiment 1 is that the gas vesicles are solidified. In this embodiment, glutaraldehyde is used for solidification. The experimental steps include:

[0154] (1) 4 mL of 25% glutaraldehyde solution was slowly added to the gas vesicles and mixed. The volume of the mixed solution was 100 mL, and the final concentration of the glutaraldehyde in the mixed solution was 1%. During this process, the uniform distribution of glutaraldehyde should be ensured to promote uniform cross-linking of the vesicle wall. After addition, the mixture was allowed to stand for 24 hours.

[0155] (2) After standing, add 8 mL of 100 mM glycine solution. After adding the glycine solution, continue to stand for 10 hours to ensure full neutralization.

[0156] (3) In order to remove unreacted glutaraldehyde and glycine, the above liquid is dialyzed for a period of not less than 24 hours. During the dialysis process, the dialysate should be replaced regularly to ensure the dialysis efficiency and removal effect.

[0157] The contrast agent obtained was tested for contrast effect, and the results were as follows: Fig. 9 As shown, Fig. 9 The effect of different mechanical indices on the contrast signal of uncured and cured gas vesicles. It can be observed from the image that the signal intensity of the uncured gas vesicles began to gradually weaken when the mechanical index (MI) increased to 0.5, and when the MI reached 0.6, the signal intensity almost dropped by half; in contrast, the cured gas vesicles can withstand higher mechanical pressure, and their signal intensity began to gradually decrease when the MI reached 0.6. This shows that the curing treatment enhances the mechanical stability of the gas vesicles, so that it can still effectively enhance the image quality and contrast under high ultrasound intensity imaging conditions.

[0158] Embodiment 23

[0159] This example investigates the conditions for solidifying gas vesicles with glutaraldehyde.

[0160] (1) The dosage of glutaraldehyde was investigated, and the final concentration of glutaraldehyde in the mixed solution was 0.5%, 0.75%, 1%, 1.5%, and 2%. Fig.10 As shown, the gas vesicles that have not been treated with glutaraldehyde show a lower ultrasound contrast signal, indicating that their stability is insufficient. In contrast, the ultrasound contrast signals of the gas vesicles treated with glutaraldehyde at different concentrations of 0.5% to 2% are significantly higher than those of the untreated group, which shows that the addition of glutaraldehyde has a significant enhancing effect on the stability of the gas vesicles, among which the gas vesicles treated with 0.5%-1.5% glutaraldehyde have better ultrasound contrast signals. With the increase of the mechanical index (MI), the ultrasound contrast signal shows a gradual downward trend. Among them, the signal decline rate of the 0.5% glutaraldehyde treatment group is the slowest, indicating that at this concentration, glutaraldehyde has the best fixation effect on gas vesicles.

[0161] In summary, the novel gastrointestinal contrast agent of the present invention has better stability and acid resistance, which can ensure that the contrast agent remains stable in the gastric cavity and provides a long-lasting imaging signal; the echogenic gastrointestinal contrast agent and the nano-scale ultrasonic contrast agent GVs work synergistically, which can make the GVs stably suspended in the solution, with bright echoes and stronger signals, and compared with ordinary echogenic gastrointestinal contrast agents, the present invention can simultaneously form B-ultrasound and contrast signals, and has good imaging performance, which provides richer diagnostic information for clinical use; the contrast agent has a delicate taste, high compliance of the subjects, and a simple and convenient brewing process, and can be brewed with drinking water at different temperatures, which reduces the waiting time of the subjects and improves the efficiency of clinical examinations.

[0162] 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 gastrointestinal ultrasound dual-display contrast agent, characterized in that: The contrast agent is a mixed solution of a biologically derived gas vesicle solution and edible grain powder particles; The volume ratio of the gas vesicle solution in the mixed solution is 0.2-0.3%, and the OD of the gas vesicle solution is 500 1.5-3.5; The concentration of edible grain powder particles in the mixed solution is 0.01-0.1 g / mL; The gas vesicles are derived from gas-vesicle-containing microorganisms; the gas-vesicle-containing microorganisms include halophilic archaea, Escherichia coli, Listeria or algae; The edible grain powder particles are prepared from edible plant powder and a binder.

2. The gastrointestinal ultrasound dual-display contrast agent according to claim 1, characterized in that: The raw materials for preparing the edible grain powder particles include 60-90 parts by weight of edible plant powder and 10-40 parts by weight of a binder; Preferably, the particle size of the gas vesicles is 100-600 nm.

3. The gastrointestinal ultrasound dual-display contrast agent according to claim 1 or 2, characterized in that: The gas vesicles are solidified gas vesicles.

4. The gastrointestinal ultrasound dual-display contrast agent according to any one of claims 1 to 3, characterized in that: The curing is glutaraldehyde curing, and the steps of the glutaraldehyde curing include: Glutaraldehyde is mixed with the gas vesicle solution, and then allowed to stand for the first time after the mixing; a glycine solution is added for neutralization, and then allowed to stand for the second time; and unreacted glutaraldehyde, glycine and reaction byproducts thereof are removed.

5. The gastrointestinal ultrasound dual-display contrast agent according to claim 4, characterized in that: The final concentration of glutaraldehyde in the mixed solution is 0.5-2%; Preferably, the first standing time is 12-36 hours.

6. The gastrointestinal ultrasound dual-display contrast agent according to claim 4 or 5, characterized in that: The second standing time is 6-12 hours.

7. The gastrointestinal ultrasound dual-display contrast agent according to any one of claims 4 to 6, characterized in that: The unreacted glutaraldehyde, glycine and their reaction byproducts are removed by dialysis.

8. The gastrointestinal ultrasound dual-display contrast agent according to any one of claims 1 to 7, characterized in that: The edible plant powder is selected from: any one of corn powder, coix seed powder, soybean powder, wheat powder or sesame powder, or a combination of at least two thereof; Preferably, the adhesive is selected from any one of lotus root starch, pectin, linseed gum, carboxymethyl cellulose or locust bean gum, or a combination of at least two thereof.

9. A method for preparing a gastrointestinal ultrasound dual-display contrast agent according to any one of claims 1 to 8, characterized in that: The method comprises: mixing edible grain powder particles and biologically derived gas vesicles with water to obtain the gastrointestinal ultrasound dual-display contrast agent.

10. Use of the gastrointestinal ultrasound dual-display contrast agent according to any one of claims 1 to 8 in the preparation of ultrasound gastrointestinal contrast examination products.

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