Photoacoustic, ultrasonic and fluorescent three-mode SonoVitamin contrast agent, preparation method thereof, application of photoacoustic, ultrasonic and fluorescent three-mode SonoVitamin contrast agent in preparation of medicine for treating kidney cancer and medicine
By developing a three-modal contrast agent composed of photoacoustic, ultrasound, and fluorescence, which is modified by renal cancer cell membrane and loaded with ICG, the limitations of the existing technology in early diagnosis and distinction of renal cancer are solved, and the efficiency and accuracy of multimodal imaging are achieved, and it has significant clinical application value.
Patent Information
- Application Number
- CN202510118011.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-24
- Publication Date
- 2025-05-30
AI Technical Summary
Existing imaging diagnostic methods have limitations in the early diagnosis of renal cancer and the distinction between renal cancer from other lesions, especially ultrasound imaging is insufficient in the detection of micro-lesion and tumor blood flow assessment, and the targeting and stability of existing contrast agents in complex tumor environments.
A photoacoustic, ultrasound, and fluorescence three-modal sonovitric contrast agent is developed, which consists of renal cancer cell membrane modified with indocyanine green (ICG)-loaded with indocyanine green (ICG). This method can improve detection sensitivity and provide high tissue resolution in a variety of imaging modes.
The contrast agent exhibits excellent multimodal imaging performance in different imaging modes, can provide accurate structural imaging, hemodynamic information and functional information of tumor cells, thereby improving the accuracy of early diagnosis and lesion localization of renal cancer, and has significant clinical application value.
Smart Images
Figure CN120053697A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of pharmaceutical technology, and particularly to a SonoVue contrast agent with photoacoustic, ultrasound, and fluorescence triple modalities, its preparation method, application in the preparation of drugs for treating renal cancer, and the drug. Background Art
[0002] Renal cancer is the most common malignant tumor of the kidney, accounting for more than 90% of kidney tumors. In recent years, with the changes in lifestyle and the development of imaging technologies, the early detection rate of renal cancer has increased to some extent, but its incidence is still rising year by year. The main pathological feature of renal cancer is the malignant hyperplasia of renal epithelial cells, accompanied by local infiltration and metastasis, especially the metastasis to the renal hilum and the lymph nodes around the kidney. Since the early symptoms of renal cancer are not obvious, patients are often diagnosed when the disease progresses to the advanced stage, resulting in many patients missing the best treatment opportunity at the initial diagnosis. Therefore, how to diagnose renal cancer at an early stage, especially how to accurately distinguish renal cancer from other lesions by imaging means, has become an urgent problem to be solved in clinical medicine.
[0003] Currently, the imaging diagnosis of renal cancer mainly relies on technologies such as CT, MRI, and ultrasound. Although these methods have made certain progress in clinical applications, there are still some limitations. For example, CT and MRI cannot completely and accurately distinguish early lesions of renal cancer, and may be limited by image resolution, tumor size, tumor morphology, and the influence of surrounding tissues; while ultrasound imaging has a relatively large lack of sensitivity in detecting micro-lesions of renal cancer, especially in evaluating tumor blood flow and micro-lesions, the ability of ultrasound is relatively weak.
[0004] In addition, in renal cancer imaging, existing contrast agents mostly rely on a single imaging mode, such as separate CT contrast enhancement or MRI contrast agents, which results in certain blind spots in the detection, localization, evaluation, and intraoperative navigation of renal cancer.
[0005] SonoVue, as a commonly used ultrasound contrast agent, has shown good effects in blood flow imaging and tissue perfusion monitoring. However, the application of SonoVue also has limitations. First, SonoVue is mainly based on the ultrasound imaging mode and is difficult to provide multi-dimensional information in a complex tumor environment, especially for the need of high-resolution anatomical imaging, blood flow monitoring, and molecular functional information at the same time, its ability is relatively limited. Second, the targeting accumulation effect of SonoVue in renal cancer is not good, and often a higher dose is required to achieve an observable imaging signal, which may increase the hidden danger of drug safety. In addition, its stability in a complex tumor microenvironment is limited and it is easily degraded in a strongly acidic or oxidative stress environment, thus affecting its diagnostic effect.
[0006] Some studies have attempted to develop multimodal imaging diagnostic contrast agents, such as contrast agents based on nanoparticle carriers, which can achieve compatibility with multiple imaging modalities through physical or chemical modifications. However, most of these contrast agents focus on optical imaging or single magnetic resonance imaging and face significant challenges in the clinical translation process, including insufficient targeting of specific tumor tissues, poor adaptability to the tumor microenvironment, and poor synergy between imaging modalities. Summary of the Invention
[0007] To solve at least one of the above technical problems, the present application proposes a SonoVue contrast agent with photoacoustic, ultrasound, and fluorescence trimodality, its preparation method, its application in the preparation of a drug for treating renal cancer, and the drug. The SonoVue contrast agent can be simultaneously applicable to multiple imaging modalities, helping to improve the detection sensitivity and having high tissue resolution.
[0008] In a first aspect, there is provided a SonoVue contrast agent with photoacoustic, ultrasound, and fluorescence trimodality, the SonoVue contrast agent comprising SonoVue microbubbles modified with renal cancer cell membranes and loaded with indocyanine green (ICG).
[0009] In a second aspect, there is provided a method for preparing the SonoVue contrast agent as described in the first aspect, comprising:
[0010] Providing renal cancer cell membranes;
[0011] Dispersing the renal cancer cell membranes in PBS to obtain a renal cancer cell membrane suspension;
[0012] Providing an ICG solution;
[0013] Providing a SonoVue solution;
[0014] Uniformly mixing the ICG solution, the renal cancer cell membrane suspension, and the SonoVue solution to obtain the SonoVue contrast agent.
[0015] In some possible embodiments, the providing of the renal cancer cell membranes includes:
[0016] Culturing renal cancer cells;
[0017] Collecting the cultured renal cancer cells;
[0018] Extracting the cell membranes of the collected renal cancer cells.
[0019] In some possible embodiments, the extracting of the cell membranes of the collected renal cancer cells includes:
[0020] Suspending the collected renal cancer cells in a hypotonic lysis solution and stirring to lyse the cell membranes of the renal cancer cells;
[0021] The lysed renal cancer cells were further disrupted by mechanical disruption, then centrifuged and the supernatant was collected;
[0022] The supernatant was separated and purified by ultrafiltration to obtain renal cancer cell membranes.
[0023] In some possible implementations, the mechanical disruption method is an ultrasonic treatment method, and the rotation speed of the centrifugal treatment is 10,000 rpm and the duration is 15 minutes.
[0024] In some possible implementations,
[0025] The method of culturing renal cancer cells comprises: culturing RENCA renal cancer cells in a culture dish using DMEM culture medium at 37°C and 5% CO 2 humidified incubator environment.
[0026] The collecting of the cultured renal cancer cells comprises: when the cultured renal cancer cells reach a confluence of 70%-80%, digesting the renal cancer cells with trypsin, and then washing at least twice with PBS to collect the renal cancer cells.
[0027] In some possible implementations,
[0028] The step of dispersing the renal cancer cell membrane in PBS to obtain a renal cancer cell membrane suspension comprises: suspending the renal cancer cell membrane in PBS and performing ultrasonic treatment to obtain a renal cancer cell membrane suspension;
[0029] The providing of the ICG solution comprises: dissolving ICG in a PBS solution and performing ultrasonic treatment to obtain the ICG solution.
[0030] In some possible embodiments, the step of uniformly mixing the ICG solution, the renal cancer cell membrane suspension and the SonoVue solution comprises:
[0031] The ICG solution and the renal cancer cell membrane suspension were added dropwise to the SonoVue solution, and stirred sufficiently to uniformly mix the ICG, renal cancer cell membrane and SonoVue.
[0032] In a third aspect, a drug for treating renal cancer loaded on the SonoVue contrast agent described in the first aspect is provided.
[0033] In a fourth aspect, a use of the SonoVue contrast agent as described in the first aspect in the preparation of a drug for treating renal cancer is provided.
[0034] The SonoVue contrast agent provided by the present application comprises SonoVue microbubbles modified with renal cancer cell membranes and loaded with indocyanine green (ICG). Among them, SonoVue has good safety and biocompatibility. As a carrier, it can reduce the side effects of the drug itself on the body and has ultrasonic imaging function; ICG, as a fluorescent dye approved by the FDA (Food and Drug Administration), has good safety and can be used as an ideal fluorescent tracer for the microbubbles and has photoacoustic imaging function; the renal cancer cell membrane has good tumor-specific targeting effect and escapes the phagocytosis of immune cells during in vivo metabolism. Its hybridization with SonoVue can endow better tumor homology and improve the targeting effect and tissue accumulation of the contrast agent. Based on this, the present application can better improve the targeting and tissue accumulation of the contrast agent and exhibit excellent multimodal imaging performance in different imaging modalities. Such a triple-modal contrast agent can simultaneously provide accurate structural imaging, hemodynamic information, and functional information of tumor cells, providing comprehensive support for the early diagnosis, lesion localization, and intraoperative navigation of renal cancer, and having significant clinical application value. Brief Description of the Drawings
[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings of the embodiments will be briefly introduced below. Obviously, the drawings in the following description only relate to some embodiments of the present application and do not limit the present application.
[0036] Figure 1 It is a flowchart of the method for preparing the SonoVue contrast agent provided by the embodiment of the present application.
[0037] Figure 2 It is a TEM image of the SonoVue contrast agent prepared in this embodiment.
[0038] Figure 3 It is a comparison chart of the particle size distributions of the SonoVue contrast agent prepared in this embodiment and ordinary SonoVue microbubbles.
[0039] Figure 4 It is a comparison chart of the potential intensities of the SonoVue contrast agent prepared in this embodiment and the SonoVue contrast agent without cell membrane encapsulation.
[0040] Figure 5 It is a comparison chart of the spectral absorption curves of the SonoVue contrast agent prepared in this embodiment and ICG.
[0041] Figure 6 It is the result of the in vitro hemolysis test of the SonoVue contrast agent prepared in this embodiment.
[0042] Figure 7The in vivo fluorescence imaging comparison diagram of SonoVue contrast agent prepared in this example and SonoVue contrast agent without cell membrane encapsulation, where (a) shows the comparison result in the form of a picture, and (b) shows the comparison result in the form of a bar chart.
[0043] Figure 8 The comparison diagram of ultrasonic contrast imaging results of SonoVue microbubbles and SonoVue contrast agent prepared in this example after being placed for different times, where (a) shows the comparison result in the form of a picture, and (b) shows the comparison result in the form of a curve graph. Specific implementation mode
[0044] To make the purpose, technical solutions and advantages of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of this application. Obviously, the described embodiments are part of the embodiments of this application, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the described embodiments of this application without creative efforts belong to the scope of protection of this application. It can be understood that, without conflict, some technical means described in the various embodiments herein may be replaced or combined with each other.
[0045] In the description of this application, references to "an embodiment" or "some embodiments" etc. mean that specific features, structures or characteristics described in conjunction with the embodiment are included in one or more embodiments of this application. Thus, the phrases "in an embodiment", "in some embodiments", "in other some embodiments", "in still other embodiments" etc. that appear in different places in this specification are not necessarily all referring to the same embodiment, but mean "one or more but not all of the embodiments", unless otherwise specifically emphasized in other ways.
[0046] Figure 1 A specific embodiment of the preparation method of SonoVue contrast agent is shown. In this embodiment, the preparation method includes the following steps (1) to (5):
[0047] (1) Provide renal cancer cell membrane.
[0048] More specifically, this step (1) includes steps (1a) to (1c):
[0049] (1a) Culture renal cancer cells. That is, culture RENCA renal cancer cells in a culture dish using DMEM medium, and the culture conditions are a humidified incubator environment at 37 °C and 5% CO 2 of.
[0050] (1b) Collect the cultured renal cancer cells. That is, when the cultured renal cancer cells reach a confluence of 70%-80%, digest the renal cancer cells with trypsin, then wash them twice with PBS (phosphate buffer solution), and collect the renal cancer cells.
[0051] (1c) Extract the cell membranes of the collected renal cancer cells. That is, suspend the collected renal cancer cells in a hypotonic lysis solution and stir to lyse the cell membranes of the renal cancer cells; further disrupt the lysed renal cancer cells using the ultrasonic treatment method (an example of mechanical disruption method), then perform centrifugation and collect the supernatant; separate and purify the supernatant by ultrafiltration to obtain renal cancer cell membranes.
[0052] Moreover, the obtained renal cancer cell membranes can be freeze-dried for later use.
[0053] (2) Suspend the renal cancer cell membranes in PBS and perform ultrasonic treatment for 10 minutes to ensure that the renal cancer cell membranes are evenly dispersed in PBS, obtaining a renal cancer cell membrane suspension.
[0054] (3) Provide an ICG (Indocyanine Green) solution. That is, weigh a certain amount of ICG and dissolve it in PBS solution, and perform ultrasonic treatment for 10 minutes to ensure complete dissolution, obtaining an ICG solution.
[0055] (4) Provide a SonoVue solution. This SonoVue solution can be purchased on the market or prepared by conventional methods, which will not be elaborated here.
[0056] (5) Drop the ICG solution and the renal cancer cell membrane suspension into the SonoVue solution and stir well to uniformly mix ICG, renal cancer cell membranes, and SonoVue. Thus, obtain the SonoVue contrast agent M SonoVue@ICG, which is a SonoVue contrast agent containing SonoVue microbubbles modified by renal cancer cell membranes and loaded with ICG, and can be used as a contrast agent for renal cancer in photoacoustic, ultrasound, and fluorescence triple-modal imaging.
[0057] It should be understood that in the embodiments of the present application, the execution order of each step should be determined according to its function and internal logic. The size of each step number does not mean the sequence of execution, and does not constitute any limitation on the implementation process of the embodiment. For example, the aforementioned steps (1) and (3) can be carried out simultaneously or in any order, and the present application does not make any limitation on this, but step (2) needs to be carried out after step (1).
[0058] Figure 2 This is the TEM image of the SonoVue contrast agent prepared in this embodiment. It can be seen from this figure that the particle size of the obtained SonoVue contrast agent is only a few microns and exists in the form of microbubbles. And,Figure 3 The particle size distributions of the SonoVue@ICG contrast agent prepared in this example and the ordinary SonoVue microbubbles are shown.
[0059] Figure 4 This is a comparison chart of the potential intensity between the SonoVue contrast agent prepared in this example and the SonoVue contrast agent without cell membrane encapsulation. The blue bars correspond to the SonoVue contrast agent prepared in this example, and the yellow bars correspond to the potential of the SonoVue contrast agent without cell membrane encapsulation. From this Figure 4 It can be seen that the potential of the nanoparticles did not change significantly before and after adding the cell membrane.
[0060] Figure 5 This is a comparison chart of the spectral absorption curves between the SonoVue contrast agent prepared in this example and ICG. The blue curve corresponds to the SonoVue@ICG contrast agent prepared in this example, and the yellow curve corresponds to ICG. From this Figure 5 It can be seen that the SonoVue contrast agent prepared in this example successfully loaded ICG.
[0061] Figure 6 This is the result of the in vitro hemolysis test of the SonoVue contrast agent prepared in this example at different concentrations. The horizontal axis represents the concentration of the SonoVue contrast agent, with the unit of μg / ml, and the vertical axis represents the hemolysis rate. From this Figure 6 It can be seen that the SonoVue contrast agent prepared in this example has no obvious destructive effect on red blood cells and has good biocompatibility.
[0062] Figure 7 This is a comparison chart of in vivo fluorescence imaging between the SonoVue contrast agent prepared in this example and the SonoVue contrast agent without cell membrane encapsulation. Among them, (a) shows the comparison result in the form of a picture, and (b) shows the comparison result in the form of a bar chart. The blue bars correspond to the SonoVue@ICG contrast agent prepared in this example, and the yellow bars correspond to the SonoVue@ICG contrast agent without cell membrane encapsulation. From this Figure 7 It can be seen that compared with the SonoVue contrast agent without cell membrane encapsulation, the SonoVue contrast agent prepared in this example has better tumor targeting effect.
[0063] Figure 8 This is a comparison chart of the contrast imaging results of ultrasound after the SonoVue microbubbles and the SonoVue contrast agent prepared in this example are placed for different times. Among them, (a) shows the comparison result in the form of a picture, and (b) shows the comparison result in the form of a curve graph. The blue curve corresponds to the SonoVue@ICG contrast agent prepared in this example, and the yellow curve corresponds to the ordinary SonoVue microbubbles. From this Figure 8It can be seen that the SonoVue contrast agent prepared in this embodiment has better stability than SonoVue microbubbles.
[0064] As can be known from the previous introduction, the SonoVue contrast agent prepared in the above embodiment contains SonoVue microbubbles modified (encapsulated) by renal cancer cell membranes and loaded with ICG. Among them, SonoVue has good safety and biocompatibility. Using it as a carrier can reduce the side effects of the drug itself on the body and has the function of ultrasonic imaging; ICG, as a fluorescent dye approved by the FDA (Food and Drug Administration), has good safety and can be used as an ideal fluorescent tracer for this microbubble and has the function of photoacoustic imaging; renal cancer cell membranes have good tumor-specific targeting effects and escape the phagocytosis of immune cells in vivo. Their hybridization with SonoVue can endow better tumor homologous targeting effects. Based on this, the therapeutic drug for renal cancer can be loaded in this SonoVue contrast agent, so as to realize the integration of the diagnosis and treatment of renal cancer tumors.
Claims
1. A photoacoustic, ultrasonic and fluorescent triple-modal SonoVue contrast agent, characterized in that: The SonoVue contrast agent contains SonoVue microbubbles modified with renal cancer cell membranes and loaded with indocyanine green (ICG).
2. A method for preparing the SonoVue contrast agent according to claim 1, characterized in that: include: Provide renal cancer cell membranes; Dispersing the renal cancer cell membrane in PBS to obtain a renal cancer cell membrane suspension; Provide ICG solution; Provide SonoVue solution; The ICG solution, the renal cancer cell membrane suspension and the SonoVue solution are uniformly mixed to obtain the SonoVue contrast agent.
3. The method according to claim 2, characterized in that The renal cancer cell membrane provided comprises: Cultivate kidney cancer cells; collecting the cultured renal cancer cells; The cell membranes of the collected renal cancer cells were extracted.
4. The method according to claim 3, characterized in that: The cell membrane of the collected renal cancer cells is extracted, comprising: The collected renal cancer cells are suspended in a hypotonic lysis solution and stirred to lyse the cell membrane of the renal cancer cells; The lysed renal cancer cells were further disrupted by mechanical disruption, then centrifuged and the supernatant was collected; The supernatant was separated and purified by ultrafiltration to obtain renal cancer cell membranes.
5. The method according to claim 4, characterized in that The mechanical disruption method is an ultrasonic treatment method, and the rotation speed of the centrifugal treatment is 10000 rpm and the duration is 15 minutes.
6. The method according to claim 3, characterized in that The method of culturing renal cancer cells comprises: culturing RENCA renal cancer cells in a culture dish using DMEM culture medium in a humidified culture box environment at 37° C. and 5% CO 2 . The collecting of the cultured renal cancer cells comprises: when the cultured renal cancer cells reach a confluence of 70%-80%, digesting the renal cancer cells with trypsin, and then washing at least twice with PBS to collect the renal cancer cells.
7. The method according to claim 2, characterized in that The step of dispersing the renal cancer cell membrane in PBS to obtain a renal cancer cell membrane suspension comprises: suspending the renal cancer cell membrane in PBS and performing ultrasonic treatment to obtain a renal cancer cell membrane suspension; The providing of the ICG solution comprises: dissolving ICG in a PBS solution and performing ultrasonic treatment to obtain the ICG solution.
8. The method according to claim 2, characterized in that: The step of uniformly mixing the ICG solution, the renal cancer cell membrane suspension and the SonoVue solution comprises: The ICG solution and the renal cancer cell membrane suspension were added dropwise to the SonoVue solution, and stirred sufficiently to uniformly mix the ICG, renal cancer cell membrane and SonoVue.
9. A drug for treating renal cancer loaded in the SonoVue contrast agent as claimed in claim 1.
10. Use of the SonoVue contrast agent as claimed in claim 1 in the preparation of a drug for treating renal cancer.