A probe liquid

Abstract

The present application relates to a kind of atherectomy intraoperative imaging device, it includes: atomizer;Probe liquid, it is contained in the atomizer;Wherein, the probe liquid is prepared by dissolving specific probe in solvent into probe stock solution, and using physiological saline to dilute probe stock solution to 1-100 μM obtain;Fluorescence in vivo imaging system is used to the image acquisition and observation of atheromatous blood vessel that atomizer continuous spray is carried out to atheromatous blood vessel.The present application can quickly, accurately deliver probe to the plaque in target blood vessel, so as to realize the stable fluorescence imaging of atheromatous plaque in the process of operation, to assist endarterectomy.The liquid droplet ejected by the atomizer used in the present application is small and uniformly distributed, and it is not easy to cause damage to fragile tissues.

Description

[0001] (This application is a divisional application of Chinese invention application 202110581320.0, filed on 2021-05-27.) Technical Field

[0002] This invention relates to the field of imaging technology for patient tissues, and more particularly to an intraoperative imaging device for atherosclerosis surgery. Background Technology

[0003] In recent years, fluorescence imaging has shown great potential in guiding surgical procedures, thus attracting widespread research interest. Besides the construction of specific fluorescent probes, safe, efficient, and rapid probe delivery systems are also a hot research topic. In vivo imaging via intravenous injection is a classic probe delivery method, but recent research indicates that during intraoperative imaging, probes can be sprayed in situ onto the target tissue surface using a spray bottle, facilitating rapid probe absorption and in-situ imaging.

[0004] Intravenous injection is inefficient because the probe takes a long time to reach the lesion, and most of the probe is removed or absorbed by other tissues before reaching the lesion. This slow delivery also increases safety risks. In-situ spraying uses a spray bottle that produces larger, unevenly distributed droplets, and the excessive spray force may damage fragile tissues. Summary of the Invention

[0005] To address the aforementioned technical issues, this invention proposes an application of in-situ spraying for imaging during atherosclerotic surgery. This method involves dissolving a specific probe in a solvent to prepare a probe stock solution, which is then diluted to 1-100 μM with physiological saline. The diluted probe solution is added to an nebulizer and continuously sprayed onto the surgically exposed atherosclerotic vessels for 10-60 seconds, followed by a resting period of 2-30 minutes. Images are then acquired and observed using a fluorescence in vivo imaging system.

[0006] As a further improvement to the application of the in-situ spray method of the present invention in imaging during atherosclerosis surgery, the structural formula of the specific probe is as follows: .

[0007] As a further improvement to the application of the in-situ spray method of the present invention in imaging during atherosclerosis surgery, the solvent is DMSO, ethyl acetate, ethanol, DMF or DCM.

[0008] As a further improvement to the application of the in-situ spray method of the present invention in imaging during atherosclerosis surgery, the concentration of the probe stock solution is 1 mM and diluted to 10 μM with physiological saline.

[0009] As a further improvement to the application of the in-situ spray method of the present invention in imaging during atherosclerosis surgery, the continuous spraying time is 15 seconds, followed by a resting time of 5 minutes.

[0010] As a further improvement to the application of the in-situ spray method of the present invention in imaging during atherosclerosis surgery, the output end of the nebulizer uses a vibrating screen to output the atomized probe solution.

[0011] As a further improvement to the application of the in-situ spray method of the present invention in imaging during atherosclerosis surgery, the mesh size of the vibrating screen is 100-500 mesh.

[0012] The present invention further proposes an in-situ spraying method, comprising the following steps: dissolving a specific probe in a solvent to prepare a probe stock solution, and diluting it to 1-100 μM with physiological saline; adding the diluted probe solution to a nebulizer and continuously spraying it onto a human organ or tissue for 10-120 seconds, followed by standing for 2-30 minutes; the structural formula of the specific probe is as follows: .

[0013] As a further improvement to the in-situ spraying method of the present invention, the solvent is DMSO, ethyl acetate, ethanol, DMF or DCM.

[0014] As a further improvement to the in-situ spraying method of the present invention, the output end of the atomizer uses a vibrating screen to output the atomized probe solution, and the mesh number of the vibrating screen is 100~500 mesh.

[0015] The advantages of this invention compared to existing technologies are: the in-situ spraying method of this invention can quickly and accurately deliver the probe to the plaque within the target blood vessel, thereby achieving stable fluorescence imaging of atherosclerotic plaques during surgery to assist in arterial endarterectomy. The nebulizer used in this invention sprays smaller and more uniformly distributed droplets, minimizing damage to fragile tissues. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be considered as a limitation on the scope of protection. For those skilled in the art, other related drawings can be obtained from these drawings without any creative effort.

[0017] Figure 1 The figure shows the experimental results of tissue imaging using the in-situ spray method provided in Example 1, including the spray pattern, duration, and settling time.

[0018] Figure 2This is the experimental result image provided in Example 2, which shows the establishment of a mouse atherosclerosis model and the imaging of atherosclerotic plaques using the in-situ spray method. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0020] This invention proposes an in-situ spray method for imaging during atherosclerotic surgery. The method involves dissolving a specific probe in a solvent to prepare a probe stock solution, which is then diluted to 1-100 μM with physiological saline. The diluted probe solution is added to a nebulizer and continuously sprayed onto the surgically exposed atherosclerotic blood vessel for 10-120 seconds, followed by a resting period of 2-30 minutes. Images are then acquired and observed using a fluorescence in vivo imaging system. This in-situ spray method can rapidly and accurately deliver the probe to the plaque within the target blood vessel, thereby achieving stable fluorescence imaging of the atherosclerotic plaque during surgery to assist in endarterectomy. The nebulizer used in this invention sprays small, evenly distributed droplets, minimizing damage to fragile tissues.

[0021] Preferably, the structural formula of the specific probe is as follows: .

[0022] The following designation, CN-PD, indicates the specific probe with the above-described structure. This specific probe exhibits excellent solvent effects, showing almost no luminescence in an aqueous phase but emitting strong green light in an organic phase. It only emits strong fluorescence in the presence of liposomes and demonstrates good selectivity by not responding to other biomolecules such as nucleic acids, proteins, and carbohydrates, with a rapid response to liposomes. This specific recognition probe is a small-molecule organic compound, exhibiting stronger penetration into the human skin.

[0023] Preferably, the solvent is DMSO, ethyl acetate, ethanol, DMF, or DCM. Among them, DMSO is non-volatile, colorless, and odorless, making it more suitable as a solvent for the above-mentioned specific probes compared to other solvents.

[0024] Preferably, the concentration of the probe stock solution is 1 mM, and it is diluted to 10 μM with physiological saline. The probe solution at this concentration exhibits stronger fluorescence.

[0025] Preferably, the continuous spraying time is 15 seconds, followed by a resting time of 5 minutes. Fluorescence imaging was performed on the test subject after continuous spraying for 0, 5, 15, 30, 60, and 120 seconds. The results showed that imaging could be performed after a continuous spraying time of 5 seconds, and stable imaging was achieved after 15 seconds. Stable imaging was achieved after continuous spraying for 15, 30, 60, and 120 seconds. In vitro imaging was performed after resting for 0.5, 1, 2, 5, and 10 minutes after continuous spraying. The results showed that stable and clear target images could be observed after resting for 5 minutes and 10 minutes.

[0026] Preferably, the output end of the nebulizer uses a vibrating screen to output the atomized probe solution. The mesh size of the vibrating screen is 100-500 mesh. By using a vibrating screen instead of a nozzle, the saline solution containing the probe is atomized into droplets with a diameter of 3μm, avoiding tissue damage caused by the high impact force of the spray bottle. Furthermore, the uniform droplets can promote good distribution and rapid tissue absorption. This method can perform intraoperative fluorescence imaging quickly and safely.

[0027] Example 1 The specific probe CN-PD was dissolved in DMSO to prepare a 1mM probe stock solution, which was then diluted to 10μM with physiological saline. The probe dilution was added to the reservoir of the Aerogen nebulizer and sprayed continuously onto the surgically exposed artery for 15 seconds, followed by 5 minutes of in vivo imaging. Images were then acquired and observed using a fluorescence in vivo imaging system. The nebulizer output used a vibrating screen with a 300-mesh sieve to atomize the probe-containing aqueous solution into a mist with a diameter of 3μm.

[0028] Example 2 The specific probe CN-PD was dissolved in DMSO to prepare a 1 mM probe stock solution, which was then diluted to 100 μM with physiological saline. The probe dilution was added to the reservoir of the Aerogen nebulizer and sprayed continuously onto the surgically exposed artery for 30 seconds, followed by a 2-minute incubation period. Images were then acquired and observed using a fluorescence in vivo imaging system. The nebulizer's output end used a vibrating screen to atomize the probe solution into a 3 μm diameter mist.

[0029] Example 3 The specific probe CN-PD was dissolved in DMSO to prepare a 1 mM probe stock solution, which was then diluted to 1 μM with physiological saline. The probe dilution was added to the reservoir of the Aerogen nebulizer and continuously sprayed onto the surgically exposed artery for 60 seconds, followed by a 30-minute incubation period. Images were then acquired and observed using a fluorescence in vivo imaging system. The nebulizer's output end used a vibrating screen to atomize the probe solution, transforming the probe-containing aqueous solution into a 3 μm diameter mist.

[0030] Experimental Example 1 To demonstrate the effectiveness of the intraoperative in-situ spray method, this study used a CN-PD small molecule lipid droplet probe with a molecular weight of 361.12 as the spray imaging reagent, and a visible light probe was selected for intraoperative fluorescence imaging of atherosclerotic plaques.

[0031] To enable rapid and safe intraoperative imaging, the inventors improved the in-situ spraying method, replacing the nozzle with a vibrating screen, such as... Figure 1 As shown in Figure a, the CN-PD solution was atomized into an aerosol using a vibrating screen, and the aqueous solution containing the probe was atomized into particles with a diameter of 3 μm, achieving rapid penetration. This improvement avoids tissue damage caused by the high impact force of the spray, and the uniform droplets promote good distribution and rapid tissue absorption.

[0032] like Figure 1 As shown in Figure b, the fat mass exposed to CN-PD aerosol (10 μM) was subjected to in vitro imaging at 0, 5, 15, 30, 60, and 120 s, respectively, to obtain differential fluorescence contrast in the adipose tissue exposed to CN-PD aerosol. There was no fluorescence at 0 s, red fluorescence was emitted at 5 s, and stable fluorescence was emitted until 15 s and beyond, indicating that the fat mass reached stable staining after 15 s of exposure to CN-PD aerosol.

[0033] In addition, the heart, liver, spleen, lungs, kidneys, intestines, A549 tumor tissue, and adipose tissue were simultaneously exposed to CN-PD aerosol for 15 seconds. Figure 1 The sub-plots (c and d) can distinguish the fat content of different organs and examine whether the fat content of different organs is normal.

[0034] like Figure 1 As shown in Figure c, heart, liver, spleen, lung, kidney, intestine, A549 tumor tissue, and adipose tissue were simultaneously exposed to CN-PD aerosol (10 μM) for 15 seconds, followed by in vitro imaging after standing for 0.5, 1, 2, 5, and 10 minutes, respectively. The imaging results are as follows. Figure 1 As shown in sub-figure d, the in vitro fluorescence intensity of the heart, liver, spleen, lung, kidney, intestine, A549 tumor tissue, and adipose tissue increased with prolonged standing time, and tended to stabilize after 5 minutes of standing. Furthermore, the in vitro fluorescence intensity varied among the organs and tissues, with the adipose tissue exhibiting the strongest fluorescence intensity and the spleen the weakest.

[0035] Experimental Example 2 The inventors selected ApoE gene-deficient C57BL / 6 mice for left carotid artery ligation and a high-fat diet to simulate an atherosclerosis model (e.g. Figure 2(See sub-figure a1 in the image). Atherosclerosis is the formation of plaques in the inner lining of arteries that can block blood flow and lead to thrombosis (as shown in Figure a1). Figure 2 As shown in sub-figure a2, sub-figure a2, from top to bottom, represents normal, atherosclerotic, and thrombus formation. During endarterectomy, surgeons need direct visualization of the exposed artery for in-situ drug delivery. The inventors have improved in-situ spraying by using a vibrating screen, enabling rapid and uniform plaque imaging of the CN-PD aerosol (e.g., ...). Figure 2 (See sub-figure a3 in the figure).

[0036] Oil Red O and H&E staining were used to test the successful establishment of atherosclerosis models, such as... Figure 2 As shown in sub-figure b. In sub-figure b, the upper figure is the blank control group that did not undergo left carotid artery ligation and a high-fat diet, and the lower figure is the experimental group that underwent left carotid artery ligation and a high-fat diet.

[0037] The process of imaging during atherosclerosis surgery in mice is as follows: Figure 2 As shown in Figure c. After modeling, the left carotid artery was exposed, and CN-PD aerosol was sprayed for 15 seconds.

[0038] like Figure 2 Subplot d shows that the top row is the control group without left carotid artery ligation and a high-fat diet, while the bottom row is the experimental group with left carotid artery ligation and a high-fat diet. Both groups were sprayed with CN-PD aerosol. As seen in the lower experimental group, after a 5-minute wait, stable and clear plaques were observed in the carotid artery, while no fluorescence reaction was observed in the control group. This plaque imaging can last for more than 2 hours, giving the surgeon ample time to perform the procedure. If the procedure is prolonged and fluorescence weakens, the probe can be repeatedly sprayed to quickly restore imaging. Plaques as small as 0.5 mm in diameter are clearly visible on the artery (e.g., Figure 2 (See sub-figure e in the image). At 5 min and 2 h after imaging, the fluorescence signal ratios (P / N) of plaque tissue and normal vascular tissue were 4.8% and 5.6%, respectively (as shown in Figure e). Figure 2 As shown in Figure f), this indicates a high signal-to-noise ratio for the fluorescence signal. Section analysis of the fluorescent portion of the artery clearly revealed fluorescently stained atherosclerotic plaques (such as...). Figure 2 (See sub-graph g in the image).

[0039] The above results confirm that the intraoperative in-situ spray method can rapidly and accurately image atherosclerotic plaques during surgery, thus assisting in arterial endarterectomy.

[0040] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A probe solution, characterized in that, The probe solution is obtained by dissolving a specific probe in a solvent to prepare a probe stock solution, and then diluting the stock solution with physiological saline to 1-100 μM; the structural formula of the specific probe is: ； The probe solution is contained in an atomizer, and the output end of the atomizer uses a vibrating screen to output the atomized probe solution. Furthermore, the atherosclerotic blood vessels continuously sprayed by the atomizer are imaged and observed using a fluorescence in vivo imaging system.

2. The probe solution according to claim 1, characterized in that: The solvent is DMSO, ethyl acetate, ethanol, DMF, or DCM.

3. The probe solution according to claim 1, characterized in that: The concentration of the probe stock solution was 1 mM, and it was diluted to 10 μM with physiological saline.

4. The probe solution according to claim 1, characterized in that: The atomizer sprays continuously for 10 to 120 seconds, then remains still for 2 to 10 minutes.

5. The probe solution according to claim 4, characterized in that, The continuous spraying time is 15 seconds, followed by a 5-minute resting time.

6. The probe solution according to claim 1, characterized in that: The vibrating screen has a mesh size of 100-500.

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