A gastrointestinal ultrasound dual-contrast agent and its application

Abstract

This invention provides a gastrointestinal ultrasound dual-contrast contrast agent and its application. 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 The concentration of the edible grain powder particles in the mixed solution is 1.5-3.5 g / mL; the gas vesicles originate from gas-bearing microorganisms; the gas-bearing microorganisms include halophilic archaea, Escherichia coli, Listeria, or algae; the edible grain powder particles are prepared from edible plant powder and binder. The contrast agent has a simple preparation method, is not prone to agglomeration, is uniformly stable, and provides good imaging results; it has better stability and acid resistance, ensuring that the contrast agent remains stable in the gastric cavity and provides a lasting imaging signal.

Description

Technical Field

[0001] This invention belongs to the fields of medicine and diagnostic technology, specifically relating to a gastrointestinal ultrasound dual-contrast agent and its application. Background Technology

[0002] Gastrointestinal diseases impose a heavy burden on people's health, making prevention and treatment urgent. Histologically, most gastrointestinal diseases are accompanied by significant changes in the morphology of the gastrointestinal wall, which can serve as a basis for diagnosis. For example, gastric ulcers erode the stomach wall, leading to changes in its morphology. Based on this principle, gastroscopy and barium meal imaging are commonly used clinically to examine gastric diseases. However, patients may experience adverse reactions such as regurgitation and stomach cramps, and some examinations involve electromagnetic radiation, resulting in low acceptance rates. In recent years, gastrointestinal filling ultrasound has emerged as a promising approach for diagnosing gastrointestinal diseases. Contrast-enhanced ultrasound imaging is non-invasive, radiation-free, low-cost, convenient, and quick, making it the most widely used medical imaging technology currently available and applicable to the diagnosis of gastrointestinal diseases.

[0003] Gastrointestinal ultrasound contrast imaging, also known as gastrointestinal distension examination, is a method that uses oral or enema contrast agents to fill the gastrointestinal tract, eliminating interference from gas and contents within the tract and thus improving the imaging effect. This allows for a clearer visualization of the gastrointestinal wall structure and lesions. Contrast agents used in gastrointestinal ultrasound contrast imaging can be divided into anechoic and echogenic types. Echogenic contrast agents have a longer retention time in the gastrointestinal tract and significantly reduce artifact interference from gas. Currently, patented oral echogenic contrast agents use grains such as rice, wheat, and sesame as the main raw materials, or incorporate microbubble ultrasound contrast agent suspensions to enhance ultrasound imaging. Lipid-based microbubbles are a widely used ultrasound contrast agent. Their particle size is usually 1-5 μm. The large particle size results in strong contrast signals. However, due to the relatively fragile physicochemical properties of phospholipid microbubbles, the preparation process of this type of contrast agent is relatively complex, with high cost and difficulty in adapting to the acidic environment of the stomach.

[0004] Current clinical applications of gastrointestinal ultrasound contrast agents present several problems: ① The preparation method is cumbersome, requiring boiling water and rapid stirring, which easily leads to coagulation and unevenness, affecting the imaging effect; ② The preparation time is relatively long, increasing patient waiting time; ③ They tend to separate into layers within a short period, failing to form a uniform paste, easily creating light spots and artifacts that affect diagnostic results; ④ The physicochemical properties of phospholipid microbubbles are relatively fragile, making the preparation process of these contrast agents complex, costly, and difficult to adapt to the acidic environment of the stomach.

[0005] Therefore, developing a convenient and inexpensive gastrointestinal contrast agent is of great significance for improving the efficiency of clinical diagnosis and treatment of gastrointestinal diseases. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a gastrointestinal ultrasound dual-contrast agent and its application. This invention combines an echogenic contrast agent with gas vesicles derived from bacteria to prepare a novel oral gastrointestinal contrast agent that synergistically enhances ultrasound contrast signals.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a gastrointestinal ultrasound dual-contrast agent, wherein the contrast agent is a mixed solution of 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, it can be 0.2%, 0.25%, or 0.3%, etc., and the OD of the gas vesicle solution is... 500 It can be 1.5-3.5, for example, 1.5, 2, 2.5, 3 or 3.5, etc.;

[0010] The concentration of edible grain powder particles in the mixed solution is 0.01-0.1 g / mL, for example, it can be 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 originate from gas-bearing microorganisms; the gas-bearing microorganisms include halophilic archaea, Escherichia coli, Listeria, or algae;

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

[0013] This invention utilizes the synergistic effect of echogenic gastrointestinal contrast agents and nanoscale ultrasound contrast agents (GVs, gas vesicles), which allows GVs to be stably suspended in the solution, resulting in bright echoes and stronger signals. Compared with ordinary echogenic gastrointestinal contrast agents, this invention can simultaneously generate ultrasound and contrast signals, providing richer diagnostic information for clinical use.

[0014] Preferably, in the raw materials for preparing the edible grain powder granules, the weight parts of edible plant powder are 60-90 parts, for example, 60 parts, 65 parts, 70 parts, 75 parts, 80 parts, 85 parts or 90 parts, and the weight parts of binder are 10-40 parts, for example, 10 parts, 15 parts, 20 parts, 25 parts, 30 parts, 35 parts or 40 parts.

[0015] In this invention, the ratio between edible plant powder and binder effectively improves the stability of the formulation, and the prepared formulation is less prone to stratification, thereby enabling more uniform imaging in the gastrointestinal tract.

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

[0017] In this invention, nanoscale GVs generally have better stability and acid resistance than microbubbles. Digestive fluids such as gastric acid may affect contrast agents, and stability and acid resistance can ensure that the contrast agent remains stable in the gastric cavity and provides a lasting imaging signal.

[0018] Preferably, the gas vesicle is a solidified gas vesicle.

[0019] To further improve the mechanical index (MI) of biologically derived gas vesicles, this invention also involves solidifying the gas vesicles. The mechanical index (MI) is a measure of ultrasonic intensity and an indicator of the gas vesicle's tolerance to ultrasound. A higher MI value indicates greater ultrasonic intensity, which may lead to gas vesicle rupture or degradation, thus affecting image quality and contrast.

[0020] Preferably, the curing is glutaraldehyde curing, and the glutaraldehyde curing step includes:

[0021] Glutaraldehyde was mixed with the gas vesicle solution and allowed to stand for the first time. Glycine solution was added to neutralize the mixture, and then it was allowed to stand for the second time to remove unreacted glutaraldehyde, glycine, and their reaction byproducts.

[0022] In this invention, glutaraldehyde-cured gas vesicles exhibit higher mechanical stability in clinical applications compared to uncured vesicles. The curing process cross-links the vesicle shell, enhancing its acoustic pressure resistance and enabling it to maintain integrity under high ultrasound intensity, thereby providing stable imaging contrast under high MI conditions.

[0023] Preferably, the final concentration of glutaraldehyde in the mixed solution is 0.5-2%, for example, it can be 0.5%, 1%, 1.5%, 2% or 2.5%; more preferably, it is 0.5-1.5%, for example, it can be 1%, 1.2%, 1.3%, 1.4% or 1.5%.

[0024] In this invention, during the mixing process of adding glutaraldehyde, it is important to ensure the uniform distribution of glutaraldehyde in order to promote uniform cross-linking of the vesicle walls.

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

[0026] In this 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 this invention, the glycine is used to neutralize glutaraldehyde.

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

[0029] Preferably, the unreacted glutaraldehyde, glycine, and their reaction byproducts are removed by dialysis.

[0030] In one specific embodiment, the dialysis treatment time is not less than 24 hours; the dialysate used in the dialysis treatment is 0.9% physiological saline; and the molecular weight cutoff (or pore size) of the filter membrane used in the dialysis treatment is 14KD.

[0031] Preferably, the edible plant powder is selected from any one or a combination of at least two of the following: corn flour, Job's tears powder, soybean flour, wheat flour, or sesame powder.

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

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

[0034] In this invention, the sieve particle size of the adhesive is 80-100 mesh, for example, it can be 80, 90 or 100, etc.

[0035] In this invention, the granular solid particles are uniform, the solution does not easily separate, and it has high stability. During ultrasound imaging, the stable suspension ensures that the hyperechoic particles are evenly distributed and do not clump together. It has a delicate taste, leading to high patient compliance. The preparation process is simple and convenient, and it can be prepared with drinking water at different temperatures, reducing patient waiting time and improving clinical examination efficiency.

[0036] In a preferred embodiment of the present invention, the edible plant powder is a combination of corn flour and Job's tears powder; the binder is lotus root powder.

[0037] In this invention, the sieved particle size of the edible grain powder 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 this invention, the particles obtained by sieving with a 0.5-1.5mm sieve dissolve quickly and uniformly, and are not prone to clumping.

[0039] In this invention, the edible grain powder granules are obtained using a granulation process. The granulation process is a wet granulation process. The wet granulation process includes: mixing edible plant powder and a binder in a specific ratio, adding a wetting agent to form a soft material; passing the soft material through a sieve to form wet granules; drying the granules and then passing them through another sieve to obtain granular solids.

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

[0041] In this invention, the drying temperature is 60-80℃, for example, it can be 60℃, 65℃, 70℃, 75℃ or 80℃, etc.

[0042] In this invention, the wetting agent is selected from: ethanol solution, starch paste or cellulose derivative.

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

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

[0045] In this invention, the gastrointestinal ultrasound contrast agent is easy to use. The prepared granular solid is mixed with water to form a solution, which is then thoroughly mixed with the gas vesicle solution before use. The dosage can be increased proportionally as needed for different applications. The gastrointestinal ultrasound contrast agent has a smooth taste, resulting in high patient compliance. The preparation process is simple and convenient, allowing for the use of drinking water at different temperatures, reducing patient waiting time and improving clinical examination efficiency.

[0046] The gas vesicles described in this invention are prepared by a method comprising the following steps: culturing halophilic archaea to isolate microorganisms containing gas vesicles from the culture medium, lysing the microorganisms, and centrifuging to collect the released gas vesicles.

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

[0048] In this invention, the lysis solution used for lysis comprises, by concentration: 8-12 mM Tris-HCl, 2-3 mM MgCl2 and 1.5-2.5 mM CaCl2, with a pH of 7.0-7.5.

[0049] In this invention, the centrifugation conditions are: 0-4℃, 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-contrast agent described in the first aspect, the method comprising: mixing edible grain powder particles and biologically derived gas vesicles with water to obtain the gastrointestinal ultrasound dual-contrast agent.

[0051] In this invention, the OD values ​​of the gas vesicles before and after dilution 500 The values ​​are shown below. OD values ​​were obtained by adding 10 mL of gas vesicle solution to 500 mL of water. 500 For example, the OD value of the gas vesicle solution before dilution. 500 Values ​​were 1.5, 2.0, 2.5, 3.0, and 3.5; the OD values ​​of the diluted solution were... 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 of OD 500 A gas vesicle solution with a value of 3.0 was added to 500 mL of water, and then 30 g of edible grain powder granules were added and mixed to obtain a gastrointestinal ultrasound dual-contrast agent.

[0053] In this invention, the OD of the gas vesicles in the gastrointestinal ultrasound dual-contrast agent... 500 Images are clear and have high contrast within the range of 0.05-0.5, with low OD. 500 Outside the designated area, it is not conducive to displaying the complete gastrointestinal boundary.

[0054] The OD of the gas vesicles described in this invention 500 The value of 0.05-0.5 refers to the OD measured after adding the gas vesicle solution to water during the preparation of gastrointestinal ultrasound dual-contrast agents. 500 The OD value is 0.05-0.5 before the addition of edible grain powder particles. 500 .

[0055] Thirdly, the present invention provides the application of the gastrointestinal ultrasound dual-contrast agent described in the first aspect in the preparation of ultrasound gastrointestinal contrast examination products.

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

[0057] This invention provides a novel gastrointestinal contrast agent, which can be used for ultrasound gastrointestinal contrast examinations, including but not limited to: gastrointestinal motility function, gastric ulcers, gastric lesions, ulcerative colitis, Crohn's disease, etc.

[0058] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all 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 a lasting imaging signal.

[0061] (2) The echo-type gastrointestinal contrast agent and the nano-scale ultrasound contrast agent GVs work synergistically. The echo-type gastrointestinal ultrasound contrast agent can enhance the contrast imaging signal intensity of GVs, and at the same time, it can make GVs stably suspended in the solution with high echo brightness. Compared with ordinary echo-type gastrointestinal contrast agents, the present invention can simultaneously form B-ultrasound and contrast signals, which provides richer diagnostic information for clinical use.

[0062] (3) The particles are uniform, the solution is not easy to separate into layers, and the stability is high. During ultrasound imaging, the stable suspension obtained after preparation allows the high echo particles to be evenly distributed, do not clump together, are not easy to empty in the stomach, and have a long imaging time.

[0063] (4) It has a delicate taste and high compliance among examinees; the brewing process is simple and convenient, and it can be brewed with drinking water at different temperatures, which reduces the waiting time of examinees and improves the efficiency of clinical examination.

[0064] (5) The gas vesicles in the contrast agent of this invention are solidified gas vesicles, which can withstand higher mechanical pressure. During detection, their signal intensity only begins to gradually decrease when the MI reaches 0.6. This indicates that the solidification treatment enhances the mechanical stability of the gas vesicles, enabling them to effectively enhance image quality and contrast even under high ultrasound intensity imaging conditions. Attached Figure Description

[0065] Figure 1These are ultrasound images of rat stomachs after rats were given a novel gastrointestinal contrast agent via gavage.

[0066] Figure 2 The results of internal sedimentation of gastrointestinal ultrasound dual-screen contrast agents prepared from different edible grain powder particles are shown in the figure.

[0067] Figure 3 Ultrasound imaging results of gastrointestinal ultrasound dual-contrast agents prepared for different edible grain powder particles.

[0068] Figure 4 The image shows the intratubular sedimentation results of gastrointestinal ultrasound dual-screen contrast agents prepared with different adhesives.

[0069] Figure 5 The images show the ultrasound imaging results of gastrointestinal ultrasound dual-contrast agents prepared from different adhesives.

[0070] Figure 6 Ultrasound imaging results of contrast agents prepared with different proportions of components.

[0071] Figure 7 The images show the ultrasound imaging results of contrast agents prepared from different gas vesicle concentrations.

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

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

[0074] Figure 10 Figure showing the results of the condition investigation for solidifying gas vesicles with glutaraldehyde. Detailed Implementation

[0075] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0076] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0077] Example 1

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

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

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

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

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

[0083] (3) Aspirate the unlysupplied microorganisms with a syringe, add PBS again, and repeat centrifugation until all microorganisms are completely lysed. Transfer the released GVs to a new centrifuge tube and store at 4°C for later use.

[0084] (B) Preparation of edible cereal flour pellets

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

[0086] (2) Mix the three ingredients in a mass percentage ratio of 40:40:20 and add 75% ethanol to make a soft material that can be kneaded into a ball and crumbled when pressed.

[0087] (3) The particles are sieved 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, the particles are passed through a 0.8 mm sieve to obtain particles with uniform particle size.

[0090] 2. Method of using oral gastrointestinal ultrasound contrast agents

[0091] The gas vesicle solution, particulate solids, and water are mixed and used for in vivo ultrasound imaging; OD 500 10 mL of a gas vesicle solution with a concentration of 3 was mixed with 30 g of edible grain powder granules and 500 mL of water to obtain a gastrointestinal ultrasound dual-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 well while adding the solid. Add 10mL of the prepared GVs (OD) 500 =3), mix well and it is ready to use. The dosage may be increased or decreased as the gastrointestinal capacity of the test subjects varies.

[0093] When used for enemas, pour 60g of the granular solid into 1200mL of water, stirring constantly until well combined; then add 20mL of the prepared GVs (OD200). 500 =3), mix well and it is ready to use.

[0094] 3. Extracorporeal ultrasound imaging

[0095] A novel gastrointestinal contrast agent was placed in the wells of a 1% agarose gel model. A linear array probe (3-11 MHz) of a Mindray Reson 7 clinical ultrasound diagnostic device was placed on one side of the agarose gel model, and imaging was performed in ultrasound contrast imaging mode. Strong and stable contrast ultrasound signals were produced. It was observed that the ultrasound contrast signals from only echogenic gastrointestinal contrast agents and only GVs were extremely weak, while the signal from the novel gastrointestinal contrast agent, a mixture of both, was significantly enhanced. Furthermore, the ultrasound signal became stronger with increasing GVs concentration.

[0096] 4. In vivo ultrasound imaging

[0097] A novel gastrointestinal ultrasound contrast agent solution was infused into the stomach of male SD rats, and in vivo ultrasound imaging was performed. The imaging results showed uniformity 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 differs from that of Embodiment 1 only 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%; and 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, with a value of 1.5. The preparation methods for gas vesicles and particulate solids are as described in Example 1.

[0100] Example 3

[0101] This embodiment provides a gastrointestinal ultrasound dual-contrast agent. The only difference between this embodiment and Embodiment 1 is that the edible grain powder granules are prepared from rice flour and lotus root powder, and the remaining steps are the same as in Embodiment 1.

[0102] Example 4

[0103] This embodiment provides a gastrointestinal ultrasound dual-contrast agent. The only difference between this embodiment and Embodiment 1 is that the edible grain powder granules are prepared from coix seed powder and lotus root powder, and the remaining steps are the same as in Embodiment 1.

[0104] Example 5

[0105] This embodiment provides a gastrointestinal ultrasound dual-contrast agent. The only difference between this embodiment and Embodiment 1 is that the edible grain powder granules are prepared from corn flour and lotus root powder, and the remaining steps are the same as in Embodiment 1.

[0106] Example 6

[0107] This embodiment provides a gastrointestinal ultrasound dual-contrast agent. The only difference between this embodiment and Embodiment 1 is that the edible grain powder granules are prepared from yam powder and lotus root powder, and the remaining steps are the same as in Embodiment 1.

[0108] Example 7

[0109] This embodiment provides a gastrointestinal ultrasound dual-contrast agent. The only difference between this embodiment and Embodiment 1 is that the edible grain powder granules are prepared from soybean powder and lotus root powder, and the remaining steps are the same as in Embodiment 1.

[0110] In the above embodiments, the effects of gastrointestinal ultrasound dual-screen contrast agents prepared from rice flour, coix seed powder, corn flour, yam powder and soybean powder on in vitro sedimentation and B-ultrasound imaging were investigated, with the effects of powder to water ratios of 4%, 6% and 8% being investigated respectively.

[0111] Figure 2 The image shows the internal sedimentation results of gastrointestinal ultrasound dual-contrast agents prepared from different edible grain powder particles. As can be seen from the image, the main factor to consider for grain powder is the uniformity of the imaging effect, while the sedimentation effect is primarily determined by the gel.

[0112] Figure 3 Ultrasound imaging results of gastrointestinal ultrasound dual-contrast agents prepared from different edible grain powder particles. As can be seen from the images, rice flour exhibits heterogeneous distribution, with significantly uneven distribution of high-signal points in its image. Yam powder and soybean powder show relatively weak imaging signals and poor image uniformity. In contrast, coix seed powder and corn powder exhibit 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-contrast agent. The only difference between this embodiment and Embodiment 1 is that the edible grain powder granules are prepared from corn flour, coix seed powder, and locust bean gum. The remaining steps are the same as in Embodiment 1.

[0115] Example 9

[0116] This embodiment provides a gastrointestinal ultrasound dual-contrast agent. The only difference between this embodiment and Embodiment 1 is that the edible grain powder granules are prepared from corn flour, coix seed powder, and guar gum. The remaining steps are the same as in Embodiment 1.

[0117] Example 10

[0118] This embodiment provides a gastrointestinal ultrasound dual-contrast agent. The only difference between this embodiment and Embodiment 1 is that the edible grain powder granules are prepared from corn flour, coix seed powder, and konjac gum. The remaining steps are the same as in Embodiment 1.

[0119] Example 11

[0120] This embodiment provides a gastrointestinal ultrasound dual-contrast agent. The only difference between this embodiment and Embodiment 1 is that the edible grain powder granules are prepared from corn flour, coix seed powder, and Prussian gum. The remaining steps are the same as in Embodiment 1.

[0121] In the above embodiments, the effects of gastrointestinal ultrasound dual-screen contrast agents prepared with different adhesives on in vitro sedimentation and B-ultrasound imaging were investigated.

[0122] Figure 4 The image shows the intratubular sedimentation results of gastrointestinal ultrasound dual-contrast agents prepared with different binders. From left to right, the images show the viscosity differences between the mixtures of five gums—locust bean gum, guar gum, konjac gum, pullulan gum, and lotus root starch—and the powder. The images show that sedimentation occurred in all test groups within 5 minutes. The sedimentation rate of the guar gum group was close to 30%, while the sedimentation rates of the other groups (except for lotus root starch) were around 20%. The sedimentation rate of the lotus root starch group was only about 4%, indicating that the lotus root starch contrast agent has the characteristics of uniform suspension and high viscosity, which can improve imaging quality and prolong gastrointestinal emptying time during the examination.

[0123] Figure 5The images show the ultrasound imaging results of gastrointestinal ultrasound dual-contrast agents prepared from different binders. From left to right, the images show the ultrasound imaging results of five gums (locust bean gum, guar gum, konjac gum, pullulan gum, and lotus root starch) before and after sedimentation with the powder. It can be observed that locust bean gum, guar gum, konjac gum, and pullulan gum exhibit numerous hyperechoic signal points in ultrasound imaging. The uneven distribution of these signal points results in coarse image quality and a lack of detail. Furthermore, during static imaging, these colloids showed signal stratification. In contrast, almost no hyperechoic signal points were detected in the lotus root starch group, and no sedimentation phenomenon was observed in the imaging results.

[0124] Example 12

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

[0126] Example 13

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

[0128] Example 14

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

[0130] Example 15

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

[0132] In the above embodiments, the effects of gastrointestinal ultrasound dual-contrast agents prepared from different proportions of edible plant powder and binder on B-mode ultrasound imaging were investigated. Specific experimental results are as follows: Figure 6 As shown.

[0133] Figure 6The images show ultrasound imaging results of contrast agents prepared with different proportions of components. It can be observed that when the mixing ratio of plant powder to binder is 80:20, the prepared contrast agent exhibits uniform and delicate image quality and high signal intensity in ultrasound imaging. In contrast, other proportions of contrast agents show high-echo signal points during imaging, and these signal points are unevenly distributed, resulting in lower overall signal intensity. Therefore, an 80:20 ratio may be the optimal ratio for achieving the best imaging effect.

[0134] Comparative Example 1

[0135] This embodiment provides a gastrointestinal ultrasound dual-contrast agent. The only difference between this embodiment and Embodiment 1 is that the contrast agent, by concentration, comprises: 0.06 g / mL edible grain powder particles, with water as the solvent, and does not contain gas vesicles.

[0136] Example 16

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

[0138] Example 17

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

[0140] Example 18

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

[0142] Example 19

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

[0144] Example 20

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

[0146] In the above embodiments, the effects of B-ultrasound and contrast imaging were investigated using gastrointestinal ultrasound dual-contrast agents with different gas vesicle concentrations. Specific experimental results are as follows: Figure 7 As shown.

[0147] Figure 7 The images show ultrasound imaging results of contrast agents prepared with different concentrations of gas vesicles. Based on the 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. Furthermore, the magnitude of the OD value has no significant impact on the ultrasound imaging signal. Therefore, provided that the contrast agent has high signal intensity and uniformity, selecting a contrast agent with an OD value above 0.16 will yield better imaging results; that is, the final OD value of the mixture obtained by dissolving the gas vesicle solution in water should be [value missing]. 500 Contrast agents with a concentration greater than 0.16 produce better imaging results.

[0148] Example 21

[0149] This embodiment investigated the effects of vesicles of different sources or types in the gastrointestinal ultrasound dual-contrast agent on the contrast agent's efficacy. The preparation steps of the gastrointestinal ultrasound dual-contrast agent were the same as in Example 1, and the sources of the vesicles used included: Halo (abbreviation of Halobacteria sp. NRC-1), BL21 (E. coli), MG1655 (E. coli), and Nissle 1917 (E. coli).

[0150] The effects of the above-mentioned dual-contrast gastrointestinal ultrasound contrast agent on B-ultrasound and contrast imaging were investigated. Specific experimental results are as follows: Figure 8 As shown.

[0151] Figure 8 The images show the ultrasound and contrast imaging results of contrast agents prepared from vesicles of different sources. When the vesicles are from Halo, the resulting contrast agent exhibits the strongest signal intensity and the best imaging homogeneity. In contrast, contrast agents prepared from vesicles of other sources show little difference in ultrasound signal performance. Therefore, in applications requiring high imaging quality and signal homogeneity, choosing Halo-derived vesicles as the contrast agent preparation material will yield superior imaging results.

[0152] Example 22

[0153] This embodiment provides a gastrointestinal ultrasound dual-contrast agent. The only difference between this embodiment and Embodiment 1 is that the gas vesicles undergo a curing process; in this embodiment, glutaraldehyde is used for curing. The experimental steps include:

[0154] (1) Slowly add 4 mL of 25% glutaraldehyde solution to the gas vesicles and mix. The volume of the mixed solution is 100 mL, and the final concentration of glutaraldehyde in the mixed solution is 1%. During this process, ensure that the glutaraldehyde is evenly distributed to promote uniform cross-linking of the vesicle walls. Let stand for 24 hours after addition.

[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 complete neutralization.

[0156] (3) To remove unreacted glutaraldehyde and glycine, the above liquid should be dialyzed for at least 24 hours. During dialysis, the dialysate should be changed regularly to ensure dialysis efficiency and removal effect.

[0157] The contrast agent was tested for its contrast effect, and the results are as follows: Figure 9 As shown, Figure 9 This study investigates the effect of different mechanical indices on the imaging signals of uncured and cured gas vesicles. The images show that the signal intensity of uncured gas vesicles begins to gradually decrease as the mechanical index (MI) increases to 0.5, and almost halves when the MI reaches 0.6. In contrast, cured gas vesicles can withstand higher mechanical pressure, and their signal intensity only begins to gradually decrease when the MI reaches 0.6. This indicates that curing enhances the mechanical stability of the gas vesicles, allowing them to effectively enhance image quality and contrast even under high-intensity ultrasound imaging conditions.

[0158] Example 23

[0159] This embodiment investigated the conditions for glutaraldehyde-cured gas vesicles.

[0160] (1) The dosage of glutaraldehyde was investigated, and the final concentrations of glutaraldehyde in the mixed solution were 0.5%, 0.75%, 1%, 1.5%, and 2%, respectively. Figure 10 As shown, untreated gas vesicles exhibited lower ultrasound contrast signals, indicating insufficient stability. In contrast, gas vesicles treated with 0.5% to 2% glutaraldehyde showed significantly higher ultrasound contrast signals than the untreated group, demonstrating that the addition of glutaraldehyde significantly enhances the stability of gas vesicles. Gas vesicles treated with 0.5%–1.5% glutaraldehyde showed even better ultrasound contrast signals. The ultrasound contrast signal gradually decreased with increasing mechanical index (MI). The 0.5% glutaraldehyde treatment group showed the slowest signal decline rate, indicating that at this concentration, glutaraldehyde had the best fixation effect on gas vesicles.

[0161] In summary, the novel gastrointestinal contrast agent of this invention exhibits better stability and acid resistance, ensuring its stability within the gastric cavity and providing a sustained imaging signal. The synergistic effect of the echogenic gastrointestinal contrast agent and the nanoscale ultrasound contrast agent GVs allows the GVs to remain stably suspended in the solution, resulting in high echo brightness and a stronger signal. Compared to ordinary echogenic gastrointestinal contrast agents, this invention can simultaneously generate ultrasound and contrast signals, providing superior imaging performance and richer diagnostic information for clinical use. Furthermore, the contrast agent has a delicate taste, high patient compliance, and a simple and convenient preparation process, allowing for dispensing with drinking water at different temperatures, reducing patient waiting time and improving clinical examination efficiency.

[0162] The applicant declares that the above description is only a specific embodiment 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 conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A gastrointestinal ultrasound dual-contrast agent, characterized in that, The contrast agent is a mixed solution of 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 It ranges from 1.5 to 3.

5. The concentration of edible grain powder particles in the mixed solution is 0.01-0.1 g / mL; The gas vesicles originate from gas-bearing microorganisms; the gas-bearing microorganisms include halophilic archaea, Escherichia coli, Listeria, or algae; The edible grain powder granules are prepared from edible plant powder and binder; In the raw materials for preparing the edible grain powder granules, the weight parts of edible plant powder are 80 parts, and the weight parts of binder are 20 parts. The edible plant powder is corn flour and / or Job's tears powder; the binder is lotus root powder; The gas vesicles have a particle size of 100-600 nm and are solidified gas vesicles.

2. The gastrointestinal ultrasound dual-contrast agent according to claim 1, characterized in that, The curing process is glutaraldehyde curing, and the glutaraldehyde curing steps include: Glutaraldehyde was mixed with a gas vesicle solution, and the final concentration of glutaraldehyde in the mixed solution was 0.5-2%. After mixing, the mixture was allowed to stand for 12-36 hours. Glycine solution was added to neutralize the mixture, and then it was allowed to stand for 6-12 hours. Unreacted glutaraldehyde, glycine and their reaction byproducts were removed by dialysis.

3. A method for preparing the gastrointestinal ultrasound dual-contrast agent as described in claim 1 or 2, characterized in that, The method includes: mixing edible grain powder particles and biologically derived gas vesicles with water to obtain the gastrointestinal ultrasound dual-contrast agent.

4. The use of the gastrointestinal ultrasound dual-contrast agent as described in claim 1 or 2 in the preparation of ultrasound gastrointestinal contrast examination products.

Images