A dual-mode acoustic contrast agent with targeting property, and a preparation method and application thereof

Abstract

The application provides a kind of dual-mode acoustic contrast agent with targeting and its preparation method and application, belong to the technical field of ultrasound contrast agent preparation. By compounding ultrasound contrast agent and fluorescent particles, a dual-mode acoustic contrast agent including ultrasound contrast agent and fluorescent probe containing DBCO structure is formed. The dual-mode acoustic contrast agent has targeting, and the DBCO group on the surface of the fluorescent probe can react with Ac4ManNAz to form an azide group enriched in tumor tissue, thereby realizing significant targeted fluorescence imaging of tumors, which is beneficial to early detection of tumors. The dual-mode acoustic contrast agent has both ultrasound imaging and targeted fluorescence imaging functions, with high imaging sensitivity and strong specificity, and has good application prospect.

Description

TECHNICAL FIELD

[0001] The application relates to a bimodal acoustic contrast agent with targeting property and a preparation method and application thereof, and belongs to the technical field of ultrasonic contrast agents. BACKGROUND

[0002] Ultrasound (ultrasonography, US) can be used as a routine imaging examination for cancer patients due to its convenience, flexibility, intuition, non-invasiveness and non-radiation. Ultrasound contrast is a more advanced diagnostic method of ultrasonography. Although traditional ultrasound contrast agents such as Sonovue have been widely used in blood flow dynamic monitoring, they lack molecular targeting and multi-modal imaging functions. Meanwhile, due to the limitations of performance parameters such as resolution, the traditional ultrasound contrast agents also have obvious limitations in early detection of tumors. In order to improve the imaging sensitivity and targeting ability, recent researches are mostly focused on composite contrast agents combining fluorescent probes and microbubble materials. However, the composite contrast agents are not ideal in terms of targeting and multi-modal, especially in the early diagnosis of tumors. SUMMARY

[0003] Therefore, the application provides a bimodal acoustic contrast agent with targeting property, which has both ultrasonic imaging and targeted fluorescent imaging functions, and high imaging sensitivity and specificity.

[0004] Specifically, the application is realized by the following scheme:

[0005] The bimodal acoustic contrast agent with targeting property comprises an ultrasonic contrast agent and a fluorescent probe containing a DBCO structure, the mass ratio of the ultrasonic contrast agent in the bimodal acoustic contrast agent is 0.2-1%, and the concentration of the fluorescent probe containing the DBCO structure in the bimodal acoustic contrast agent is 2-8 μg / ml.

[0006] The above scheme uses the ultrasonic contrast agent and the fluorescent probe containing the DBCO as active components of the contrast agent, uses the azido group of the tumor metabolic marker as a target, realizes specific active targeting of the tumor through the biological orthogonal "click chemistry" reaction between the DBCO and the azido group, has the dual-mode imaging function of ultrasonic contrast and fluorescent imaging, reduces non-specific signal interference in the diagnosis and imaging process, and can significantly improve the sensitivity and specificity of early diagnosis of tumors. The preparation process is simple, does not require complex purification and genetic modification processes, and is safe.

[0007] Further, as a preferred:

[0008] The fluorescent probe containing the DBCO structure is Cy5-DBCO.

[0009] The ultrasonic contrast agent is sulfur hexafluoride microbubbles for injection.

[0010] The mass ratio of the ultrasound contrast agent in the bimodal acoustic contrast agent is 0.5-0.6%.

[0011] The concentration of the fluorescent probe containing the DBCO structure in the bimodal acoustic contrast agent is 4-5 μg / ml.

[0012] The bimodal acoustic contrast agent with targeting property is in the form of emulsion, and the particle size is 3-5 μm.

[0013] Meanwhile, the applicant also provides a preparation method of the bimodal acoustic contrast agent, and the steps are as follows:

[0014] Step one, the fluorescent probe containing the DBCO structure is dissolved in physiological buffered saline (PBS), and a mixed solution is obtained by fully shaking.

[0015] Preferably, in the mixed solution, the concentration of the fluorescent probe containing the DBCO structure is 10-20 μM.

[0016] Step two, the ultrasound contrast agent is added to the mixed solution obtained in step one, and the obtained uniform emulsion is the bimodal acoustic contrast agent with targeting property after shaking until the ultrasound contrast agent is completely dissolved.

[0017] In the preparation process, the fluorescent probe with good water solubility and selective reaction ability with azide molecules is introduced on the basis of the ultrasound contrast agent. The fluorescent probe has a click chemistry structural unit with biological orthogonal reaction ability, can combine with functional sugar molecules metabolized in tumor tissues, realizes selective enrichment through a click reaction, and thus significantly enhances the imaging signal of the tumor region and improves the sensitivity and specificity of early diagnosis.

[0018] Therefore, the bimodal acoustic contrast agent with targeting property can be used in the application of in vitro ultrasound contrast imaging, in vivo ultrasound contrast imaging and targeted fluorescence imaging. In particular, when applied to the targeted fluorescence imaging of tumors, the bimodal acoustic contrast agent can target and image the tumor state and early tumor formation, realize early diagnosis of tumors and real-time monitoring during surgery.

[0019] Before the in vitro ultrasound contrast imaging, in vivo ultrasound contrast imaging and targeted fluorescence imaging, in addition to intravenous injection of the bimodal acoustic contrast agent with targeting property to the imaging and imaging objects, Ac4ManNAz can also be intravenously injected to the contrast imaging objects in advance.

[0020] During the in vitro ultrasound contrast imaging, in vivo ultrasound contrast imaging and targeted fluorescence imaging, ultrasound stimulation can be assisted for imaging.

[0021] Compared with the prior art, the present application has the following advantages:

[0022] 1) The bimodal acoustic contrast agent of the present application introduces a DBCO group modified fluorescent probe on the basis of ultrasound contrast agent, which realizes functional expansion through simple physical mixing, without changing the original structure or stability of the ultrasound contrast agent, and without gene modification or complex purification process, which is simple in technology and easier to scale up production; at the same time, based on the marketed commercial ultrasound contrast agent such as sulfur hexafluoride microbubble for injection, the safety of which is also clinically endorsed.

[0023] 2) The fluorescent probe introduced in the present application can realize high specificity binding with pre-administered Ac4ManNAz labeled tumor tissue through bio-orthogonal click reaction (DBCO-Azide), forming a tumor metabolic marker azido group, which significantly improves the imaging selectivity and fluorescence signal intensity of the tumor region. The multi-modal imaging and precise targeting of the present application solves the problem of lack of molecular targeting of traditional ultrasound contrast agents.

[0024] 3) The composite contrast agent has dual functions of ultrasound contrast and fluorescence imaging, and is oriented to diagnosis in application, which is simpler in technology and closer to clinical needs, avoiding the complexity of development of gene engineering drugs, and can realize preoperative diagnosis, intraoperative navigation and postoperative monitoring of the whole process of visual support, which has broad clinical application potential. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application.

[0026] Figure 1 The fluorescence microscope images of the bimodal contrast agent of the present application (the scales are all 10 μm),

[0027] BF (Bright Field, bright field): refers to the imaging mode of the sample under ordinary white light illumination,

[0028] Cy5: refers to the fluorescence imaging mode of the sample irradiated by excitation light with a wavelength of about 650 nm, and collected at about 670 nm;

[0029] Figure 2 DLS spectrum of the bimodal contrast agent SDC and Sonovue of the present application;

[0030] Figure 3 Transmission electron microscope images of the bimodal contrast agent SDC and Sonovue of the present application (the scales are all 500 nm);

[0031] Figure 4 Toxicity test of the bimodal contrast agent SDC and Sonovue of the present application on Huh-7 cells;

[0032] Figure 5 The in vivo imaging of the dual-mode contrast agent SDC of the present application is shown in the following figure.

[0033] Figure 6 The uptake of the dual-mode contrast agent SDC of the present application by Huh-7 under ultrasound stimulation is shown in the following figure.

[0034] Figure 7 The in vivo imaging of the dual-mode contrast agent SDC of the present application is shown in the following figure.

[0035] Figure 8 The ultrasound contrast of tumors under different contrast conditions within 3 min is shown in the following figure.

[0036] Figure 9 The intensity-time curve of the ultrasound contrast of tumors by the dual-mode contrast agent SDC of the present application is shown in the following figure.

[0037] Figure 10 The ultrasound contrast of tumors of six mice under in vivo imaging instrument within 18 h and 30 h is shown in the following figure.

[0038] Figure 11 The ultrasound contrast of tumors of the dual-mode contrast agent SDC of the present application within 18~66 h is shown in the following figure. DETAILED DESCRIPTION

[0039] In order to make the technical problems to be solved by the present application, the technical solutions and beneficial effects clearer and more apparent, the technical solutions in the embodiments of the present application will be further described in detail below with reference to the drawings in the embodiments of the present application. It should be understood that the specific embodiments described herein are only used to explain the present application, and are not used to limit the technical solutions of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0040] For the convenience of introducing the scheme, the ultrasound contrast agent in the present embodiment uses sulfur hexafluoride microbubbles for injection, and specifically, commercially available Sonovue can be used. The following will be specifically described.

[0041] The present embodiment provides a dual-mode acoustic contrast agent with targeting property, which comprises Sonovue and Cy5-DBCO (CAS No.: 1564286-24-3), and is a uniform light blue emulsion.

[0042] The preparation process of the above-mentioned dual-mode acoustic contrast agent is as follows:

[0043] (1) Take 4 μL of Cy5-DBCO (20 μM) and dissolve it in 5 mL of phosphate buffered saline (PBS), shake well to form a uniform light blue fluorescent solution.

[0044] (2) Immediately add the above fluorescent solution to the Sonovue lyophilized powder bottle (25 g), shake quickly and continuously for about 20 s until the lyophilized powder is completely dissolved and a stable, uniform light blue emulsion liquid is formed, which is a bimodal acoustic contrast agent, denoted as Sonovue-DBCO-Cy5, abbreviated as SDC.

[0045] The emulsion liquid prepared by the above method is identified and performance tested, and the results are as follows:

[0046] 1) Identification: The obtained emulsion liquid is added dropwise on a glass slide (left image in Figure 1 ), and after fixation, it is observed under a fluorescence microscope, and the results are shown in the right image in Figure 1 : The microbubbles successfully combined with the fluorescent label, indicating that the bimodal acoustic contrast agent SDC of the application is successfully prepared, and the fluorescent label is stably present (lower right image). The conventional contrast agent Sonovue has no fluorescent effect.

[0047] 2) Morphological characterization: The obtained emulsion liquid is subjected to particle size and morphological characterization.

[0048] The dynamic light scattering (DLS) results are shown in Figure 2 : The average particle size of SDC is about 3.3 μm, and PDI = 0.032, with a concentrated distribution. The average particle size of the conventional contrast agent Sonovue is about 3.1 μm, and PDI = 0.109.

[0049] Figure 3 The transmission electron microscopy (TEM) image further verifies that the bimodal acoustic contrast agent SDC prepared in the application is a uniform spherical particle (right image), which is consistent with the DLS detection results; the bimodal acoustic contrast agent SDC has good structural stability at the nanometer / micrometer scale (e.g. Figure 3 500 nm scale in ).

[0050] 3) Safety evaluation

[0051] Take the effect on the cell survival rate of Huh-7 cells as an example, this part carries out a cytotoxicity test on the prepared bimodal contrast agent SDC, the process is as follows:

[0052] Huh-7 cells are inoculated in a 96-well plate at a concentration of 10 × 10 3 cells / mL, and cultured for 24 h. Then different concentrations (10 6 ~10 8The cell culture medium was replaced by the bimodal contrast agent solution of the application (0.5 ml, 0.5 mg / ml) and incubated for 6 h. After incubation, the cells were washed with PBS and the cell survival rate was evaluated by the standard CCK-8 method.

[0053] By Figure 4 It can be seen that even at a concentration of 10 8 The cell survival rate of the bimodal contrast agent SDC of the application is still high (about 96%) at a concentration of 0.5 mg / ml, indicating that the bimodal contrast agent provided by the application is relatively safe for cells; the survival rate of the conventional contrast agent Sonovue is less than 90% at the same concentration.

[0054] 4) Contrast imaging of the bimodal contrast agent SDC of the application under ultrasound in vitro

[0055] 1 ml of the prepared bimodal contrast agent SDC and the conventional contrast agent Sonovue were respectively dropped into the prepared gel mold, and the contrast imaging over time was observed under the ultrasound contrast mode, and the results are shown in Figure 5 As shown in the figure, the contrast imaging stability of the bimodal contrast agent SDC of the application is similar to that of the conventional contrast agent Sonovue, and the clearance at 2 h is even slightly better than that of the conventional contrast agent.

[0056] 5) Targeting imaging ability and ultrasound-promoted uptake experiment of the bimodal contrast agent SDC of the application

[0057] To study the tumor-targeting fluorescence imaging ability and ultrasound-assisted effect of the bimodal contrast agent SDC of the application, Huh-7 cells (2x10 5 cells / dish) were inoculated in a confocal culture dish and incubated with a culture medium containing Ac4ManNAz (4 μL, 80 μM / mL) for 24 h to introduce an azido group (Az).

[0058] Subsequently, the groups were treated as follows:

[0059] ① SDC group (without introducing Az);

[0060] ② Az-introduced + DBCO-Cy5 group

[0061] ③ Az-introduced + bimodal contrast agent of the application;

[0062] ④ Az-introduced + bimodal contrast agent of the application + ultrasound (US, 1 MHz, 1.5 W / cm 2 , 1 min).

[0063] After incubation for 20 min, the cells were washed with PBS and stained with Hoechst 33342, and then observed under a confocal fluorescence microscope. Figure 6 ).

[0064] In Figure 6 which Hoechst 33342 channel shows blue fluorescence representing cell nucleus; Cy5 channel shows red fluorescence representing fluorescent probe or contrast agent combined by click chemistry; Merge is superimposed image for showing the relationship of fluorescent localization.

[0065] The results show that: ① group (i.e. Figure 6 the first column in FIG. 6) only shows weakly dispersed punctate red fluorescence; ② and ③ groups (i.e. Figure 6 the second column and the third column in FIG. 6) can both observe red fluorescence signal, and the signal shows continuous linear pattern along the cell membrane profile, indicating that the specific enrichment of contrast agent on target cells is realized by strain-promoted azide-alkyne cycloaddition (SPAAC) reaction; and ④ group (i.e. Figure 6 the fourth column in FIG. 6) under the assistance of ultrasound, the fluorescence signal is significantly enhanced, and the red signal is more concentrated in the cytoplasmic region, indicating that the endocytosis of cells to the contrast agent is further promoted by ultrasound stimulation. The results confirm that the bimodal contrast agent of the application has good targeted imaging ability, and ultrasound treatment can synergistically enhance its uptake efficiency in tumor cells.

[0066] 6) Application of the bimodal contrast agent SDC of the application in in vivo targeted fluorescence imaging

[0067] To further verify the targeted uptake ability of the bimodal contrast agent SDC of the application in live animals and the ultrasound-enhanced effect, 6-8-week-old nude mice were selected to construct a subcutaneous tumor model for in vivo imaging experiments.

[0068] Each mouse was subcutaneously injected with 2×10 6 tumor cells (Huh-7), and about 2 weeks after the formation of obvious tumor nodules, the mice were divided into the following three groups (3 mice in each group):

[0069] Group A: no Ac4ManNAz treatment, no US;

[0070] Group B: continuous tail vein injection of Ac4ManNAz (4 μL, 80 μM / mL, dissolved in 100 μL PBS), no US;

[0071] Group C: continuous tail vein injection of Ac4ManNAz (4 μL, 80 μM / mL, dissolved in 100 μL PBS), and receiving ultrasound stimulation (1 MHz, 1.5 W / cm 2 , 2 min) after tail vein injection of the bimodal contrast agent SDC of the application.

[0072] The three groups of mice were all tail vein injected with the bimodal contrast agent SDC of the application on the third day, and then placed in the small animal live imaging system (IVIS) for fluorescence imaging analysis at different time points.

[0073] The experimental results are shown in Figure 7

[0074] The tumor region of group A has no obvious fluorescence signal, indicating that the contrast agent is difficult to selectively accumulate when there is no click reaction or targeting structure.

[0075] The tumor site of group B has obvious fluorescence accumulation, which proves that the click reaction of DBCO and Az groups in the tumor tissue makes the contrast agent target accumulation.

[0076] Group C shows the strongest fluorescence signal, indicating that on the basis of the click reaction, the ultrasound stimulation further enhances the enrichment degree of the contrast agent in the tumor site.

[0077] This experiment fully demonstrates that the dual-mode contrast agent SDC of the application has good tumor targeting and in vivo fluorescence imaging capability, and the targeted uptake efficiency can be further improved by ultrasound means.

[0078] 7) Application of the dual-mode contrast agent of the application in in vivo ultrasound contrast imaging

[0079] To verify the ultrasound contrast capability of the dual-mode contrast agent SDC of the application in vivo and its correlation with tumor targeting, the following small animal imaging experiment was carried out.

[0080] The established subcutaneous tumor nude mouse model was divided into three groups (3 in each group):

[0081] Group A (control group): tail vein injection of traditional Sonovue;

[0082] Group B (non-targeting experimental group): injection of the dual-mode contrast agent SDC of the application, without Ac4ManNAz treatment;

[0083] Group C (targeting experimental group): injection of the dual-mode contrast agent SDC of the application, and continuous tail vein injection of Ac4ManNAz (4 μL, 80 μM / mL, dissolved in 100 μL PBS) for 2 days before contrast.

[0084] All mice were immediately placed under the ultrasound instrument (ultrasound frequency 1 MHz, power 1.5 W / cm 2 ) after injection of the contrast agent, and images were collected using the conventional ultrasound contrast mode, and the imaging intensity and duration of the tumor region of each group were recorded in real time.

[0085] The experimental results are shown in Figure 8

[0086] The imaging signal of group A is short and decays rapidly after about 1 minute;

[0087] Group B has a moderate intensity of contrast signal, but the duration is limited;​​

[0088] Group C shows higher signal intensity and longer duration (3 minutes non-clearance), indicating that the click reaction induced by Ac4ManNAz makes the contrast agent SDC more easily enriched in the tumor area, thus prolonging its retention time in the local area and enhancing the ultrasound contrast effect.

[0089] Quantitative statistical analysis results (see Figure 9 ) further confirm the above observations:

[0090] (1) The signal of the clinically commonly used non-targeted contrast agent Sonovue group decays rapidly, indicating that it lacks specific retention ability;

[0091] (2) The SDC group itself does not have an active targeting function, and its signal maintenance time is slightly better than that of the Sonovue group, but it still gradually decreases over time;

[0092] (3) The Ac4ManNAz + SDC group realizes specific tumor binding through the metabolic labeling-click chemistry targeting strategy, so it shows significant and persistent signal enrichment after initial uptake, and maintains a stable high-intensity signal throughout the observation period.

[0093] The above comparison results show that the targeting strategy of the Ac4ManNAz pre-labeling combined with SDC designed in this study can significantly enhance the specific retention and signal persistence of the contrast agent in the tumor site.

[0094] 8) Application of the dual-mode contrast agent SDC in early tumor detection

[0095] To evaluate the performance of the dual-mode contrast agent in the early imaging of tumors, the established subcutaneous tumor model was randomly divided into two groups, with six nude mice in each group:

[0096] Group A (fluorescence group): tail vein injection of the dual-mode contrast agent SDC of the application, which can simultaneously realize targeted fluorescence imaging;

[0097] Group B (control group): tail vein injection of traditional ultrasound contrast agent Sonovue.

[0098] At the time of model establishment, each nude mouse was subcutaneously injected with 2x10 6 tumor cells (Huh-7). After 6 hours of tumor cell inoculation, the tumor cells were basically stable and adhered to form a group, and imaging monitoring was started according to the time points.

[0099] Group A imaging procedure: after 6 hours of inoculation, the dual-mode contrast agent SDC of the application was injected into the tail vein, and small animal fluorescence in vivo imaging (IVIS system) was performed every 12 hours after injection until obvious fluorescence signal aggregation was observed in the tumor-bearing area.

[0100] B. Imaging procedure: 6 hours after inoculation, SonoVue was injected via tail vein and ultrasound imaging was performed at the same time point (every 12 hours) until contrast filling signal was observed in the tumor region.

[0101] The experimental results are as follows:

[0102] Figure 10 The in vivo fluorescence imaging results of group A at 18 hours and 30 hours after tumor inoculation are shown. All six mice showed consistent imaging characteristics, and obvious fluorescence signal in the tumor region was observed at 30 hours. Figure 11 The dynamic changes of ultrasound contrast in the tumor region from 18 hours to 66 hours after tumor inoculation are shown. It can be seen that the contrast agent can be clearly detected in the tumor until 66 hours.

[0103] The above results prove that the dual-mode contrast agent SDC of the present application can detect tumors earlier than the traditional ultrasound contrast agent SonoVue, especially in the early cell mass stage, and can achieve high sensitivity detection, providing strong support for early clinical diagnosis.

[0104] The above-described embodiments only express several possible implementation manners of the present application, and the description is relatively specific and detailed, but it should not be understood as a limitation on the scope of the patent of the present application. The embodiments also do not limit the protection scope in the claims of the present application. For ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, and equivalent implementations or changes made without departing from the present application should be included in the present application.

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. The ultrasound contrast agent is an injectable sulfur hexafluoride microbubble. 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 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.

4. 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.

5. 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.

6. The preparation method according to claim 5, characterized in that: In step one, the concentration of the fluorescent probe containing the DBCO structure in the mixed solution is 10~20 μM.

7. The application of the targeted bimodal acoustic contrast agent of claim 1 in the preparation of reagents for in vitro ultrasound contrast imaging, in vivo ultrasound contrast imaging, or targeted fluorescence imaging, wherein, before imaging, the targeted bimodal acoustic contrast agent and Ac4ManNAz are respectively administered intravenously to the subject to be imaged or imaged.

8. The application according to claim 7, characterized in that: The imaging or imaging process is combined with ultrasound stimulation.

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