Targeting bimodal acoustic contrast agent as well as preparation method and application thereof

Abstract

The invention provides a bimodal acoustic contrast agent with targeting property as well as a preparation method and application thereof, and belongs to the technical field of ultrasonic contrast preparations. An ultrasonic contrast agent and fluorescent particles are subjected to composite construction to form the bimodal acoustic contrast agent comprising the ultrasonic contrast agent and a fluorescent probe containing a DBCO structure. The bimodal acoustic contrast agent has targeting property, and a DBCO group modified on the surface of the fluorescent probe can be subjected to click reaction with Ac4ManNAz to form an azide group enriched in a tumor tissue, so that remarkable targeting fluorescence imaging of a tumor is realized, and early discovery of the tumor is facilitated. The bimodal acoustic contrast agent has both ultrasonic imaging and targeted fluorescence imaging functions, is high in imaging sensitivity and strong in specificity, and has a good application prospect.

Description

Technical Field

[0001] This application relates to a targeted dual-modal acoustic contrast agent, its preparation method, and its application, belonging to the field of ultrasound contrast agent technology. Background Technology

[0002] Ultrasound (US) is a routine imaging examination for cancer patients due to its simplicity, flexibility, intuitiveness, non-invasiveness, and lack of radiation. Contrast-enhanced ultrasound is a more advanced diagnostic method within ultrasound examination. While traditional contrast agents such as SonoVe are widely used for dynamic blood flow monitoring, they lack molecular targeting and multimodal imaging capabilities. Furthermore, limitations in performance parameters such as resolution also significantly limit the early detection of tumors using traditional contrast agents. To improve imaging sensitivity and targeting capabilities, recent research has focused on composite contrast agents combining fluorescent probes and microbubble materials. However, the resulting composite contrast agents have not shown ideal results in terms of targeting and multimodal capabilities, particularly in the early diagnosis of tumors. Summary of the Invention

[0003] In view of this, this application provides a targeted dual-modal acoustic contrast agent that combines ultrasound imaging and targeted fluorescence imaging functions, with high imaging sensitivity and strong specificity.

[0004] Specifically, this application is implemented through the following scheme: A targeted dual-modal acoustic contrast agent includes an ultrasound contrast agent and a fluorescent probe containing a DBCO structure. The mass ratio of the ultrasound contrast agent in the dual-modal acoustic contrast agent is 0.2-1%, and the concentration of the fluorescent probe containing the DBCO structure in the dual-modal acoustic contrast agent is 2-8 μg / ml.

[0005] The aforementioned scheme uses an ultrasound contrast agent and a DBCO-containing fluorescent probe as the active components of the contrast agent, targeting azide groups that are tumor metabolic markers. Through a bioorthogonal "click chemistry" reaction between DBCO and the azide groups, it achieves specific active targeting of the tumor. It combines the dual-modal imaging capabilities of ultrasound contrast imaging and fluorescence imaging, reducing non-specific signal interference during diagnosis and imaging, and significantly improving the sensitivity and specificity of early tumor diagnosis. The preparation process is simple, requiring no complex purification or gene modification processes, and it has high safety.

[0006] Furthermore, as a preferred option: The fluorescent probe containing the DBCO structure is Cy5-DBCO.

[0007] The ultrasound contrast agent is sulfur hexafluoride microbubbles for injection.

[0008] The mass ratio of the ultrasound contrast agent to the dual-modal acoustic contrast agent is 0.5-0.6%.

[0009] The concentration of the fluorescent probe containing the DBCO structure in the dual-modal acoustic contrast agent is 4~5 μg / ml.

[0010] The aforementioned targeted dual-modal acoustic contrast agent is in emulsion form with a particle size of 3-5 μm.

[0011] Meanwhile, the applicant also provides a method for preparing the above-mentioned dual-modal acoustic contrast agent, the steps of which are as follows: Step 1: Dissolve the fluorescent probe containing the DBCO structure in physiological buffered saline (PBS) and shake thoroughly to obtain a mixed solution.

[0012] Preferably, the concentration of the fluorescent probe containing the DBCO structure in the mixed solution is 10~20 μM.

[0013] Step 2: Add the ultrasound contrast agent to the mixed solution obtained in Step 1, and shake until the ultrasound contrast agent is completely dissolved. The resulting homogeneous emulsion is the targeted dual-modal acoustic contrast agent.

[0014] The above preparation process introduces a fluorescent probe with good water solubility and selective reactivity with azide molecules into the ultrasound contrast agent. This fluorescent probe has a click chemical structural unit with bioorthogonal reaction capability, which can bind to functional carbohydrate molecules that are metabolic markers in tumor tissue. Through the "click reaction", selective enrichment is achieved, thereby significantly enhancing the imaging signal of the tumor area and improving the sensitivity and specificity of early diagnosis.

[0015] Therefore, the aforementioned targeted dual-modal acoustic contrast agents can be used in in vitro ultrasound contrast imaging, in vivo ultrasound contrast imaging, and targeted fluorescence imaging. In particular, when applied to targeted fluorescence imaging of tumors, these dual-modal acoustic contrast agents can target and image tumor status and early tumor formation, enabling early tumor diagnosis and real-time intraoperative monitoring.

[0016] Before the aforementioned in vitro ultrasound contrast imaging, in vivo ultrasound contrast imaging, and targeted fluorescence imaging, in addition to intravenously injecting the aforementioned targeted dual-modal acoustic contrast agent into the imaging subject, Ac4ManNAz can also be injected intravenously into the imaging subject beforehand.

[0017] In the aforementioned in vitro ultrasound contrast imaging, in vivo ultrasound contrast imaging, and targeted fluorescence imaging processes, ultrasound stimulation can be used to assist imaging.

[0018] Compared with the prior art, the present invention has the following advantages: 1) The dual-modal acoustic contrast agent of this invention introduces a fluorescent probe modified with a DBCO group on the basis of an ultrasound contrast agent. The function is extended through simple physical mixing without changing the original structure or stability of the ultrasound contrast agent, or without gene modification or complex purification process. The technology is simple and easier to scale up. At the same time, it is based on commercially available ultrasound contrast agents such as injectable sulfur hexafluoride microbubbles, which also provides clinical endorsement for its safety.

[0019] 2) The fluorescent probe introduced in this application can achieve highly specific binding with pre-administered Ac4ManNAz-labeled tumor tissue in vivo via a bioorthogonal click reaction (DBCO-Azide), forming an azide group targeting tumor metabolic markers, significantly improving the imaging selectivity and fluorescence signal intensity of the tumor region. This application's multimodal imaging and precise targeting solve the problem of the lack of molecular targeting in traditional ultrasound contrast agents.

[0020] 3) This composite contrast agent has the dual functions of ultrasound contrast imaging and fluorescence imaging. When used, it is diagnostically oriented, the technology is simpler and closer to clinical needs, and it avoids the complexity of developing gene-engineered drugs. It can realize full-process visualization support for preoperative diagnosis, intraoperative navigation and postoperative monitoring, and has broad clinical application potential. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application.

[0022] Figure 1 The images show fluorescence micrographs of the dual-modal contrast agent used in this application (scale bar is 10 μm). BF (Bright Field): refers to the imaging mode of a sample under ordinary white light illumination. Cy5: refers to irradiating the sample with excitation light at a wavelength of approximately 650 nm and acquiring its fluorescence imaging mode at approximately 670 nm; Figure 2 The DLS spectra of the dual-modal contrast agent SDC and SonoVe in this application are shown. Figure 3 Transmission electron microscopy images of the dual-modal contrast agents SDC and SonoVe in this application (scale bar is 500 nm). Figure 4 This study tested the toxicity of the dual-modal contrast agent SDC and SonoVe to Huh-7 cells. Figure 5 This study compares the time-stability of the dual-modal contrast agent SDC in this application with that of SonoVe under ultrasound imaging. Figure 6This is a comparison diagram showing the uptake of the dual-modal contrast agent SDC by Huh-7 under ultrasound stimulation in this application; Figure 7 This is a representation of the in vivo fluorescence imaging of the dual-modal contrast agent SDC of this application; Figure 8 The effects of different contrast-enhanced ultrasound conditions on tumor imaging within 3 minutes; Figure 9 The image shows the intensity-time curve of the dual-modal contrast agent SDC for tumor ultrasound imaging in this application. Figure 10 The results of contrast-enhanced ultrasound imaging of tumors in six mice at 18h and 30h after tumor bearing were obtained using an in vivo imaging system. Figure 11 This is a comparison of the dual-modal contrast agent SDC used in this application with the ultrasound contrast imaging of tumors 18-66 hours after tumor formation. Detailed Implementation

[0023] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the technical solutions in the embodiments of this application will be further described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit the technical solutions of this application. All other embodiments obtained by those skilled in the art based on the embodiments in this application without creative effort are within the scope of protection of this application.

[0024] For ease of explanation, this embodiment uses injectable sulfur hexafluoride microbubbles as the ultrasound contrast agent, specifically commercially available SonoVe. The details are explained below.

[0025] This embodiment provides a targeted dual-modal acoustic contrast agent, including SonoVe and Cy5-DBCO (CAS No.: 1564286-24-3), which is a uniform pale blue emulsion liquid.

[0026] The preparation process of the above-mentioned dual-modal acoustic contrast agent is as follows: (1) Weigh 4 μL of Cy5-DBCO (20 μM), dissolve it in 5 mL of phosphate-buffered saline (PBS), shake well to mix, and form a uniform light blue fluorescent solution.

[0027] (2) Immediately add the above fluorescent solution to the Sonovue lyophilized powder bottle (25g), shake rapidly and continuously for about 20s until the lyophilized powder is completely dissolved and forms a stable, uniform light blue emulsion, which is the dual-modal acoustic contrast agent, denoted as Sonovue-DBCO-Cy5, abbreviated as SDC.

[0028] The emulsion prepared by the above method was identified and its performance was tested. The results are as follows: 1) Identification: The resulting emulsion is dropped onto a glass slide ( Figure 1 (Left image in the image), after fixation, was observed under a fluorescence microscope, and the results are as follows. Figure 1 As shown in the right figure, the microbubbles successfully bound to the fluorescent label, indicating that the dual-modal acoustic contrast agent SDC of this application was successfully prepared and the fluorescent label is stably present (bottom right figure). In contrast, the conventional contrast agent SonoVe showed no fluorescence.

[0029] 2) Morphological characterization: The obtained emulsion liquid was characterized by particle size and morphology.

[0030] Dynamic light scattering (DLS) results are as follows Figure 2 As shown, SDC has an average particle size of approximately 3.3 μm, a PDI of 0.032, and a concentrated distribution. In contrast, the conventional contrast agent SonoVil has an average particle size of approximately 3.1 μm and a PDI of 0.109.

[0031] Figure 3 Further verification using transmission electron microscopy (TEM) images confirmed that the dual-modal acoustic contrast agent SDC prepared in this application consists of uniform spherical particles (right figure), which is consistent with the DLS detection results; this dual-modal acoustic contrast agent SDC is spherical at the nanometer / micrometer scale (e.g., Figure 3 The 500nm scale in the model exhibits good structural stability.

[0032] 3) Security assessment Taking the effect on the cell viability of Huh-7 cells as an example, this section describes the cytotoxicity test of the prepared dual-modal contrast agent SDC, as follows: Huh-7 cells were used at a concentration of 10 × 10⁻⁶ 3 Cells were seeded at a concentration of [number] cells / mL in 96-well plates and cultured for 24 h. Then, cells were seeded with different concentrations (10 [units]...). 6 ~10 8 The original cell culture medium was replaced with the dual-modal contrast agent culture medium of this application (cells / ml). After incubation for 6 h, the cells were washed with PBS, and cell viability was assessed using the standard CCK-8 assay.

[0033] Depend on Figure 4 It can be seen that even 10 8 At a cell / ml concentration, the cell survival rate of the dual-modal contrast agent SDC in this application is still very high (approximately 96%), indicating that the dual-modal contrast agent provided in this application is relatively safe for cells; at the same concentration, the survival rate of the conventional contrast agent SonoVe is less than 90%.

[0034] 4) Imaging of the dual-modal contrast agent SDC under in vitro ultrasound in this application 1 ml of the prepared dual-modal contrast agent SDC and the conventional contrast agent SonoVe were respectively dropped into the prepared gel mold. The contrast imaging over time was observed in ultrasound contrast mode. The results are as follows: Figure 5 As shown, even after 2 hours, it can still be observed that the contrast imaging stability of the dual-modal contrast agent SDC of this application is similar to that of the conventional contrast agent SonoVe, and the clearance at 2 hours is even slightly better than that of the conventional contrast agent.

[0035] 5) Targeting imaging capability and ultrasound-enhanced uptake experiment of the dual-modal contrast agent SDC of this application. To investigate the tumor-targeting fluorescence imaging capability and ultrasound-assisted effect of the dual-modal contrast agent SDC proposed in this application, Huh-7 cells (2×10⁻⁶) were used. 5 (1 atom / dish) were inoculated into confocal culture dishes and incubated with medium containing Ac4ManNAz (4 μL, 80 μM / mL) for 24 h to introduce azide groups (Az).

[0036] The groups were then processed as follows: ① SDC group (Az not introduced); ② Introduce the Az+DBCO-Cy5 group ③ Introducing Az+ and the dual-modal contrast agent SDC of this application; ④ Introduction of Az+ dual-modal contrast agent of this application + ultrasound (US, 1 MHz, 1.5 W / cm) 2 (1 min).

[0037] After incubation for 20 min, each group was washed with PBS and stained with Hoechst 33342 for nuclear staining, followed by observation under a confocal fluorescence microscope. Figure 6 ).

[0038] exist Figure 6 In the image, the Hoechst 33342 channel displays blue fluorescence, representing the cell nucleus; the Cy5 channel displays red fluorescence, representing fluorescent probes or contrast agents that bind via click chemistry; and the Merge image is an overlay image used to show the fluorescence localization relationship.

[0039] The results showed that: Group ① (i.e. Figure 6 The first column showed only weak, scattered, punctate red fluorescence; groups ② and ③ (i.e., Figure 6 In the second and third columns, red fluorescence signals were observed, exhibiting a continuous linear pattern along the cell membrane contour, indicating that the contrast agent achieved specific enrichment on target cells through strain-promoted azido-acetylene cycloaddition (SPAAC) reaction; while in group ④ ( Figure 6(In the fourth column) Under ultrasound-assisted imaging, the fluorescence signal was significantly enhanced, with the red signal more concentrated in the cytoplasm, indicating that ultrasound stimulation further promoted the endocytosis of the contrast agent by cells. This result confirms that the dual-modal contrast agent of this application has good targeted imaging capabilities, and that ultrasound treatment can synergistically enhance its uptake efficiency in tumor cells.

[0040] 6) Application of the dual-modal contrast agent SDC in in vivo targeted fluorescence imaging. To further verify the targeted uptake ability and ultrasound enhancement effect of the dual-modal contrast agent SDC in living animals, subcutaneous tumor models were constructed using 6-8 week old nude mice for in vivo imaging experiments.

[0041] Each mouse was injected subcutaneously with 2×10 6 After approximately two weeks, once tumor cells (Huh-7) formed obvious tumor nodules, the mice were divided into three groups (n=3 per group): Group A: Ac4ManNAz will not be processed, and US will not be accepted; Group B: Continuous tail vein injection of Ac4ManNAz (4 μL, 80 μM / mL, dissolved in 100 μL PBS), US not accepted; Group C: Continuous tail vein injection of Ac4ManNAz (4 μL, 80 μM / mL, dissolved in 100 μL PBS), followed by ultrasound stimulation (1 MHz, 1.5 W / cm²) after tail vein injection of the dual-modal contrast agent SDC of this application. 2 (2 min).

[0042] All three groups of mice were injected with the dual-modal contrast agent SDC of this application via the tail vein on day 3, and were subsequently placed in a small animal in vivo imaging system (IVIS) for fluorescence imaging analysis at different time points.

[0043] The experimental results are as follows Figure 7 As shown: The absence of obvious fluorescence signals in the tumor area of ​​Group A indicates that contrast agents are difficult to selectively accumulate in the absence of click reactions or targeted structures.

[0044] The presence of significant fluorescence accumulation in the tumor sites of group B demonstrates that the click reaction between DBCO and the Az groups in the tumor tissue enables the contrast agent to target and accumulate.

[0045] Group C showed the strongest fluorescence signal, indicating that ultrasound stimulation further enhanced the enrichment of contrast agent at the tumor site, building upon the click response.

[0046] This experiment fully demonstrates that the dual-modal contrast agent SDC of this application has good tumor targeting and in vivo fluorescence imaging capabilities, and that the targeted uptake efficiency can be further improved by ultrasound.

[0047] 7) Application of the dual-modal contrast agent of this application in in vivo ultrasound contrast imaging To verify the in vivo ultrasound imaging capability of the dual-modal contrast agent SDC and its correlation with tumor targeting, the following small animal imaging experiment was conducted.

[0048] The established subcutaneous tumor nude mouse model was divided into three groups (3 mice in each group): Group A (control group): Traditional Sonove was injected via tail vein; Group B (non-targeted experimental group): injected with the bimodal contrast agent SDC of this application, and did not receive Ac4ManNAz treatment; Group C (targeted experimental group): The bimodal contrast agent SDC of this application was injected, and Ac4ManNAz (4 μL, 80 μM / mL, dissolved in 100 μL PBS) was injected into the tail vein for 2 consecutive days before the contrast.

[0049] All mice were immediately placed in an ultrasound apparatus (1 MHz ultrasound frequency, 1.5 W / cm²) after injection of the contrast agent. 2 Under conventional ultrasound contrast imaging mode, images were acquired, and the imaging intensity and duration of each tumor region were recorded in real time.

[0050] The experimental results are as follows Figure 8 As shown: The imaging signal in Group A is brief, lasting for about 1 minute before rapidly decaying; Group B showed moderate-intensity contrast signals, but with limited duration; Group C showed higher signal intensity and longer duration (no clearance for 3 minutes), indicating that the Ac4ManNAz-induced click response makes the contrast agent SDC more easily enriched in the tumor area, thereby prolonging its local retention time and enhancing the ultrasound imaging effect.

[0051] Quantitative statistical analysis results (see) Figure 9 This further confirms the above observation: (1) The signal of the commonly used non-targeted contrast agent SonoVe rapidly decayed, indicating that it lacked specific retention ability; (2) The SDC group itself does not have an active targeting function. Although its signal maintenance time is slightly better than that of the SonoVe group, it still gradually decreases over time. (3) The Ac4ManNAz + SDC group achieved specific tumor binding through a metabolic labeling-click chemistry targeting strategy, thus exhibiting significant and persistent signal enrichment after initial uptake and maintaining a stable high-intensity signal throughout the observation period.

[0052] The above comparison results show that the targeting strategy designed in this study, which combines Ac4ManNAz prelabeling with SDC, can significantly enhance the specific retention and signal persistence of contrast agents at the tumor site.

[0053] 8) Application of the dual-modal contrast agent SDC in the early detection of tumors (as per this application) To evaluate the performance of the dual-modal contrast agent of this application in early tumor imaging detection, the established subcutaneous tumor model was randomly divided into two groups, with six nude mice in each group: Group A (fluorescence group): The dual-modal contrast agent SDC of this application was injected into the tail vein, which can simultaneously achieve targeted fluorescence imaging; Group B (control group): Traditional ultrasound contrast agent SonoVe was injected via tail vein.

[0054] During model establishment, each nude mouse was subcutaneously injected with 2×10⁻⁶ mg / L. 6 One tumor cell (Huh-7). Six hours after inoculation, the tumor cells were basically stable and adhered together, and imaging monitoring began at time points.

[0055] Group A imaging procedure: Six hours after inoculation, the dual-modal contrast agent SDC of this application is injected into the tail vein, and small animal fluorescence in vivo imaging (IVIS system) is performed every 12 hours after injection until obvious fluorescence signal aggregation is observed in the tumor-bearing area.

[0056] B-group imaging procedure: Six hours after inoculation, SonoVe is injected via the tail vein, and contrast-enhanced ultrasound imaging is performed at the same time points (every 12 hours) until contrast agent filling signals are observed in the tumor area.

[0057] The experimental results are as follows: Figure 10 The results of in vivo fluorescence imaging of group A at 18 and 30 hours after tumor bearing are shown. All six mice exhibited consistent imaging characteristics, with obvious fluorescence signals observed in the tumor-bearing area at 30 hours. Figure 11 The study demonstrated the dynamic changes in the tumor region via contrast-enhanced ultrasound over 18 to 66 hours, showing that contrast agent filling within the tumor could be clearly detected up to 66 hours later.

[0058] The above results demonstrate that the dual-modal contrast agent SDC of this application can detect tumors earlier than the traditional ultrasound contrast agent SonoVe, especially in the early cell cluster stage, achieving high-sensitivity detection and providing strong support for early clinical diagnosis.

[0059] The above-described embodiments are merely illustrative of several feasible implementations of the present invention, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of the present invention, nor are the embodiments intended to limit the scope of protection in the claims of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the concept of the present invention. All equivalent implementations or changes that do not depart from the present invention should be included in the technology of the present invention.

Claims

1. A targeted dual-modal acoustic contrast agent, characterized in that: The mixture includes an ultrasound contrast agent and a fluorescent probe containing a DBCO structure, wherein the mass ratio of the ultrasound contrast agent to the dual-modal acoustic contrast agent is 0.2-1%, and the concentration of the fluorescent probe containing the DBCO structure in the dual-modal acoustic contrast agent is 2-8 μg / ml.

2. The targeted dual-modal acoustic contrast agent according to claim 1, characterized in that: The fluorescent probe containing the DBCO structure is Cy5-DBCO.

3. The targeted dual-modal acoustic contrast agent according to claim 1, characterized in that: The ultrasound contrast agent is sulfur hexafluoride microbubbles for injection.

4. The targeted dual-modal acoustic contrast agent according to claim 1, characterized in that: The mass ratio of ultrasound contrast agent to dual-modal acoustic contrast agent is 0.5-0.6%, and the concentration of fluorescent probe containing DBCO structure in dual-modal acoustic contrast agent is 4-5 μg / ml.

5. The targeted dual-modal acoustic contrast agent according to claim 1, characterized in that: The targeted dual-modal acoustic contrast agent is in emulsion form with a particle size of 3-5 μm.

6. A method for preparing the dual-modal acoustic contrast agent according to claim 1, characterized in that, The steps are as follows: Step 1: Dissolve the fluorescent probe containing the DBCO structure in physiological buffer saline and shake thoroughly to obtain a mixed solution; Step 2: Add the ultrasound contrast agent to the mixed solution obtained in Step 1, and shake until the ultrasound contrast agent is completely dissolved. The resulting homogeneous emulsion is the targeted dual-modal acoustic contrast agent.

7. The preparation method according to claim 6, characterized in that: In step one, the concentration of the fluorescent probe containing the DBCO structure in the mixed solution is 10~20 μM.

8. The application of the targeted dual-modal acoustic contrast agent of claim 1 in in vitro ultrasound contrast imaging, in vivo ultrasound contrast imaging, or targeted fluorescence imaging.

9. The application according to claim 8, characterized in that: Before imaging, the subjects are intravenously injected with a targeted dual-modal acoustic contrast agent and Ac4ManNAz.

10. The application according to claim 8, characterized in that: The imaging or imaging process is combined with ultrasound stimulation.

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