An ultrasound contrast agent targeting tumor thrombosis, its preparation method and application

Abstract

This invention discloses an ultrasound contrast agent targeting tumor thrombosis, its preparation method, and its application. The contrast agent comprises: a perfluorinated carbon gas microbubble core, an amphiphilic film-forming material shell, and a targeting portion attached to the shell surface. The targeting portion specifically binds to tumor thrombosis-related markers selected from tissue factor, podoplanin, fibrin, activated platelet membrane glycoproteins GPIIb / IIIa, or P-selectin. In vitro experiments show that the adhesion number of the targeted microbubbles of this invention to tumor thrombosis is 10 times that of the clinical non-targeted microbubbles SonoVue, with a residual rate of 78% at arterial shear rates. In vivo imaging shows that the targeted microbubbles have a diagnostic sensitivity of 91.7%, specificity of 83.3%, and AUC of 0.96, significantly superior to SonoVue (sensitivity 41.7%, AUC 0.52). This invention achieves specific ultrasound diagnosis of tumor thrombosis, exhibiting unexpected technical advantages compared to existing clinical non-targeted microbubbles, and possesses clinical translational value.

Description

Technical Field

[0001] This invention relates to the fields of biomedical engineering and molecular imaging, specifically to an ultrasound contrast agent targeting tumor thrombosis, its preparation method, and its application in diagnosis. Background Technology

[0002] Cancer-associated thrombosis (CAT) is one of the most common complications in patients with malignant tumors, with an incidence rate as high as 4-20%, and is the second leading cause of death after cancer progression. Clinical studies have shown that tumor thrombosis and ordinary thrombosis are fundamentally different in pathophysiology: tumor thrombosis contains surviving tumor cells, which directly activate the coagulation cascade by secreting procoagulant substances such as tissue factor (TF) and podoplanin. Accurately distinguishing between tumor thrombosis and ordinary thrombosis is crucial for clinical treatment decisions—patients with tumor thrombosis require combined anticoagulation and antitumor therapy, while misdiagnosis as ordinary thrombosis will delay antitumor treatment and lead to a worse prognosis.

[0003] Currently, non-targeted microbubble contrast agents (such as SonoVue, Definity, etc.) used in routine clinical ultrasound examinations have the following technical defects: (1) Lack of specificity: Non-targeted microbubbles are visualized through passive blood flow pools and cannot distinguish between tumor thrombi and ordinary thrombi. Both appear as intravascular filling defects on ultrasound images, making differential diagnosis difficult. Existing clinical studies show that the diagnostic sensitivity of non-targeted microbubbles for tumor thrombi is only 40-60%, and the specificity is less than 50%, close to the level of random guessing. (2) Insufficient binding ability: Non-targeted microbubbles rely on physical interception or passive aggregation in areas of blood stasis and are easily washed away under the action of blood flow shear force, resulting in weak adhesion to thrombi. Under arterial blood flow conditions (shear rate > 500 s), -1 (3) Short diagnostic window: The retention time of non-targeted microbubbles at the thrombus site is usually less than 5 minutes, which cannot meet the time requirements of clinical operations (such as multi-slice scanning and doctor's judgment).

[0004] While some literature reports ultrasound contrast agents targeting thrombi or tumors, these studies have not validated the specific pathological entity of "tumor thrombi." More importantly, existing research lacks head-to-head comparisons with clinically available non-targeted microbubbles, failing to demonstrate the clinical superiority of targeted design and lacking quantitative diagnostic efficacy indicators (such as sensitivity, specificity, and AUC value). Therefore, developing an ultrasound contrast agent that can specifically identify tumor thrombi and has significantly superior diagnostic efficacy compared to clinically available non-targeted microbubbles has significant clinical value. Summary of the Invention

[0005] The purpose of this invention is to provide a targeted ultrasound contrast agent that can specifically identify tumor thrombi, and whose diagnostic efficacy (including sensitivity, specificity and AUC value) is significantly better than that of clinical non-targeted microbubbles, thus solving the technical problem that existing ultrasound contrast agents cannot distinguish between tumor thrombi and ordinary thrombi and have low sensitivity in diagnosing tumor thrombi.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides an ultrasound contrast agent targeting tumor thrombosis, comprising: a gas microbubble core containing perfluorinated carbon gas, a microbubble shell coating the gas microbubble core and composed of an amphiphilic film-forming material, and a targeting portion specifically binding to tumor thrombosis-related markers connected to the outer surface of the shell; wherein the tumor thrombosis-related markers are selected from tissue factor, podoplanin, fibrin, activated platelet membrane glycoprotein GPIIb / IIIa, or P-selectin.

[0008] Specifically, it includes: (a) a gas microbubble core containing perfluorinated carbon gas; (b) a microbubble shell covering the gas microbubble core, composed of an amphiphilic film-forming material; and (c) a targeting portion covalently connected to the outer surface of the shell, specifically binding to tumor thrombosis-related markers; wherein the tumor thrombosis-related markers are selected from: tissue factor, podoplanin, fibrin, activated platelet membrane glycoprotein GPIIb / IIIa, or P-selectin.

[0009] Its working principle is as follows: Targeted microbubbles, through their surface-displayed targeted portions, specifically bind to biomarkers highly expressed in tumor thrombi, achieving active anchoring. Under physiological and pathological blood flow shear forces, targeted microbubbles can stably bind to the thrombus surface, while non-targeted microbubbles (such as SonoVue) cannot bind effectively. Through ultrasound contrast imaging, targeted microbubbles generate persistent, high-contrast enhanced signals at the tumor thrombus site, thereby achieving specific diagnosis of tumor thrombi.

[0010] Preferably, the targeting portion is covalently connected to the microbubble shell layer via a DSPE-PEG connecting arm.

[0011] Preferably, the perfluorocarbon gas is selected from SF6, C3F8, or C4F. 10 .

[0012] Preferably, the average diameter of the microbubbles is 1-5 μm.

[0013] The present invention also provides a method for preparing the targeted tumor thrombosis ultrasound contrast agent, comprising the following steps:

[0014] (1) Couple the film-forming material with the target portion to obtain a target ligand-film-forming material conjugate;

[0015] (2) The coupling material from step (1) is mixed with additional film-forming material in proportion, dissolved in an organic solvent, and rotary evaporated to form a thin film;

[0016] (3) Add buffer solution to hydrate the film, and prepare microbubbles by mechanical vibration under a perfluorinated carbon gas atmosphere;

[0017] (4) Purify to obtain targeted microbubbles.

[0018] The third aspect of the present invention also provides the application of the above-mentioned ultrasound contrast agent targeting tumor thrombosis in the preparation of reagents for ultrasound imaging diagnosis of tumor thrombosis.

[0019] The beneficial effects of this invention are:

[0020] Compared with existing clinical non-targeted microbubbles (represented by SonoVue), this invention has the following beneficial effects:

[0021] (1) Significantly improves the binding capacity of tumor thrombi: In vitro flow models show that the number of adhesions of the targeted microbubbles of this invention to tumor thrombi is 8-10 times that of SonoVue, and at an arterial blood flow shear rate (500 s⁻¹), the binding capacity is significantly improved. -1 The residual rate under ( ) exceeds 75%, while SonoVue is less than 15%.

[0022] (2) For the first time, ultrasound differential diagnosis of tumor thrombus and ordinary thrombus was achieved: The signal intensity of the targeted microbubbles of this invention on tumor thrombus is 3-7 times that on ordinary thrombus (SonoVue cannot distinguish it, the ratio is about 1.05), and the area under the receiver operating characteristic curve (AUC) reaches 0.96, while SonoVue is only 0.52.

[0023] (3) Significantly improve diagnostic sensitivity and specificity: With peak signal enhancement >8dB as the positive judgment standard, the sensitivity of the microbubble targeted by this invention is 91.7%, the specificity is 83.3%, and the accuracy is 87.5%; SonoVue is only 41.7%, 50.0%, and 45.8%.

[0024] (4) Extended diagnostic imaging window: The signal half-life of the targeted microbubbles at the thrombus site is extended to 11.5 minutes (3.2 minutes for SonoVue), providing sufficient time for clinical operation.

[0025] (5) Seamless integration with existing clinical procedures: The preparation process is mature, and the usage method is exactly the same as that of non-targeted microbubbles, without the need to modify ultrasound equipment. Attached Figure Description

[0026] Figure 1This is a schematic diagram of the structure of the targeted microbubbles of the present invention. Detailed Implementation

[0027] The following detailed description, in conjunction with embodiments, illustrates an ultrasound contrast agent targeting tumor thrombosis, its preparation method, and its application, but these should not be construed as limiting the scope of protection of this invention.

[0028] Example 1: Preparation and characterization of anti-tissue factor (TF) monoclonal antibody targeted microvesicles

[0029] The materials and reagents used include:

[0030] Target component: Anti-human tissue factor monoclonal antibody (purchased from American Diagnostica);

[0031] Phospholipids DSPC (distearylphosphatidylcholine), DSPE-PEG2000-NHS, cholesterol;

[0032] Perfluoropropane (C3F8, purity ≥99%)

[0033] Clinical control group SonoVue (Bracco, Italy; contains SF6 microbubbles, a marketed non-targeted contrast agent);

[0034] The structure of microbubbles is shown in the attached figure. Figure 1 As shown, the specific preparation steps are as follows:

[0035] (1) Ligand conjugation: DSPE-PEG2000-NHS was dissolved in PBS buffer (0.1M, pH 8.0), and anti-TF antibody (molar ratio 1:1) was added. The reaction was carried out overnight at 4°C. After the reaction, the unbound antibody was removed by purification using a 10kDa ultrafiltration tube, and the DSPE-PEG-antibody conjugate was obtained by lyophilization. The conjugation efficiency was verified by SDS-PAGE to be 72±6%.

[0036] (2) Microbubble preparation: Weigh 80 mg DSPC, 15 mg cholesterol, and 5 mg DSPE-PEG-antibody (molar ratio approximately 80:15:5), dissolve in 5 mL chloroform, and rotary evaporate (40℃, 80 rpm) to form a uniform film. Add 10 mL Tris buffer (10 mM, pH 7.4, containing 5% glycerol), hydrate at 60℃ for 30 minutes to obtain a liposome suspension. Transfer to a 20 mL vial, fill with perfluoropropane gas, and shake using a VialMix mechanical shaker for 45 seconds. After standing and separating, collect the upper milky white microbubble layer.

[0037] (3) Purification: Centrifuge at low speed (300×g, 3 minutes) to remove unencapsulated free antibodies and broken liposomes. Collect the floating microbubble layer by flotation, wash 3 times with PBS (300×g, 3 minutes each time), and finally resuspend in PBS and store at 4°C.

[0038] The particle size of the prepared microbubbles was determined using a dynamic light scattering particle size analyzer (Malvern Nano ZS): average diameter 2.5 ± 0.6 μm, polydispersity index (PDI) 0.12 ± 0.03. The microbubble concentration was counted using a hemocytometer: (2.8 ± 0.5) × 10⁻⁶. 8 antibody concentration / mL. Antibody conjugation efficiency, as determined by FITC-labeled secondary antibody and flow cytometry, was 72 ± 6%. Antibody density, as determined by quantitative ELISA, was 4200 ± 800 antibodies / microbubble.

[0039] Example 2: In vitro targeted binding experiment (compared with SonoVue)

[0040] The steps for constructing tumor thrombosis mimics are as follows:

[0041] (1) Cell culture: Human pancreatic cancer cells PANC-1 (ATCC CRL-1469) were cultured in DMEM medium containing 10% fetal bovine serum to the logarithmic growth phase. Flow cytometry confirmed that PANC-1 cells highly expressed tissue factor (TF), with a positive rate >95%.

[0042] (2) Preparation of tumor thrombosis mimicry: PANC-1 cells (1×10⁻⁶) were used to prepare the tumor thrombosis mimicry. 6 Platelet count / mL) and platelet-rich plasma (platelet count >300×10⁻⁶) 9 Mix ( / L) at a 1:1 volume ratio, add thrombin (final concentration 2U / mL), and incubate at 37°C for 30 minutes to form a thrombus-like clot containing tumor cells. The positive control group consisted of platelet-rich plasma without PANC-1 cells + thrombin (a common thrombus mimic). The negative control group consisted of PBS-coated culture dishes.

[0043] Static binding assay: Tumor thrombus mimicry, ordinary thrombus mimicry, and blank control were coated onto the bottom of a 96-well plate (100 μL per well, incubated overnight at 4°C). Targeted microbubbles or SonoVue (concentration 1 × 10⁻⁶) from Example 1 were added to each well. 8 The plate was incubated at 37°C for 30 minutes with 100 μL of PBS buffer per well (200 μL each time). The plate was washed three times with PBS buffer, with the plate placed on a shaker (100 rpm, 1 minute) during each wash. The number of adhered microbubbles in each high-power field (200×) was counted under an inverted microscope, with five fields counted per well. The experimental results are shown in Table 1.

[0044] Table 1

[0045]

[0046] The above results indicate that the number of targeted microvesicles adhering to tumor thrombi was 10.1 times that of SonoVue (342 vs 34, p < 0.001, t-test). The adhesion ratio of targeted microvesicles to tumor thrombi versus ordinary thrombi was 4.0 (342 / 86), while the ratio of SonoVue was 1.10 (34 / 31, p > 0.05, no significant difference).

[0047] Flow-binding experiment: A parallel-plate flow chamber system (GlycoTech, USA) was used to simulate physiological blood flow conditions. A tumor thrombus simulator was coated onto a glass slide and mounted in the flow chamber. A physiological flow rate (shear rate 100 s⁻¹) was applied. -1 Arterial flow velocity (shear rate 500 s) -1 ) and shear rate at narrow points (1500 s) -1 ) Infuse microbubble suspension (1×10 8 The flow rate was 100 microbubbles / mL for 2 minutes, followed by perfusion with PBS buffer at the same flow rate for 2 minutes. The number of microbubbles adhering to the field of view before and after perfusion was counted under a microscope, and the residual rate (after perfusion / before perfusion × 100%) was calculated. The experimental results are shown in Table 2.

[0048] Table 2

[0049]

[0050] The above results show that at 500 s -1 At arterial shear rate, the residual rate of targeted microbubbles (78%) was 6.5 times that of SonoVue (12%).

[0051] In summary, the targeted microbubbles of Example 1 exhibit a high degree of specificity in binding to tumor thrombi, significantly outperforming the clinical non-targeted microbubbles SonoVue under both static and flowing conditions, and maintaining stable binding under physiological and pathological blood flow shear forces.

[0052] Example 3: In vivo ultrasound imaging experiment (compared with SonoVue)

[0053] The steps for constructing an animal model are as follows:

[0054] (1) Tumor thrombosis model: BALB / c nude mice (female, 6-8 weeks old, weighing 18-22g, purchased from Beijing Vital River) were subcutaneously inoculated with PANC-1 cells (1×10⁻⁶). 6 Each individual was suspended in 100 μL of Matrigel. Tumor volume was increased to 500 ± 50 mm². 3At approximately 2-3 weeks, a mixture of human thrombin (10 U / animal) and fibrinogen (200 μg / animal) (total volume 100 μL) was injected via the tail vein to induce thrombosis within the tumor tissue. Histological sections confirmed that PANC-1 tumor cells were embedded in the fibrin network within the thrombus.

[0055] (2) Conventional thrombosis model (control): Healthy BALB / c nude mice (of the same age and weight as above) were injected into the femoral vein with a mixture of human thrombin (10U / mouse) and fibrinogen (200μg / mouse) to induce lower extremity venous thrombosis.

[0056] The experiment involved 3 groups of 6 animals each:

[0057] Group A (Experimental Group): Injected with targeted microbubbles as described in Example 1 (dose 1×10⁻⁶). 8 (One microbubble / animal, administered via tail vein injection);

[0058] Group B (positive control): SonoVue was injected (at the recommended dose as directed in the instructions, via tail vein injection).

[0059] Group C (negative control): Injected with an equal volume of PBS buffer;

[0060] The ultrasound imaging procedure was as follows: A Vevo 2100 high-resolution small animal ultrasound imaging system (FUJIFILM VisualSonics) was used, with a probe frequency of 18MHz, Contrast Mode, Mechanical Index (MI) = 0.10, Gain of 30dB, and Dynamic Range of 45dB. Ultrasound images were continuously acquired for 15 minutes after microbubble injection via the tail vein (30-second acquisitions at 0.5, 1, 2, 3, 5, 10, and 15 minutes post-injection). The region of interest (ROI, thrombus site) was manually delineated (approximately 2-5 mm in area). 2 The signal intensity (in dB) was measured using Vevo LAB software, and the enhancement factor was calculated based on the difference between the baseline signal before injection. Simultaneously, the signal intensity of the surrounding normal tissue was measured, and the contrast-to-noise ratio (CNR) was calculated: CNR = (Signal Intensity_ROI - Signal Intensity_Background) / Background Standard Deviation.

[0061] The experimental results are shown in Table 3-4.

[0062] Table 3

[0063] Tumor thrombosis model:

[0064]

[0065] The results show that the peak signal enhancement of the targeted microbubbles (16.8 dB) is 5.6 times that of SonoVue (3.0 dB) (p < 0.001). The CNR of the targeted microbubbles (5.8) meets the clinical diagnostic requirements (usually ≥ 3.0), while the CNR of SonoVue (0.9) does not.

[0066] Table 4

[0067] Standard thrombosis model:

[0068]

[0069] Diagnostic efficacy analysis: The peak signal enhancement > 8 dB was used as the criterion for positive tumor thrombosis (this threshold was determined by the implementer's operating characteristic curve). The results are shown in Table 5.

[0070] Table 5

[0071]

[0072] Plot the receiver operating characteristic (R℃) curve and calculate the area under the curve (AUC):

[0073] Group A (targeted microbubbles): AUC = 0.96 (95% confidence interval: 0.91–1.00);

[0074] Group B (SonoVue): AUC = 0.52 (95% confidence interval: 0.38-0.66);

[0075] The AUC of the targeted microbubbles (0.96) was significantly higher than that of SonoVue (0.52), and the AUC of SonoVue was close to 0.5 (random guess level).

[0076] In summary, Example 1 demonstrated that targeted microbubbles achieved specific ultrasound imaging of tumor thrombosis in live animals, with diagnostic efficacy (sensitivity 91.7%, specificity 83.3%, AUC 0.96) significantly superior to the first-line clinical non-targeted contrast agent SonoVue, and possesses clear clinical translational value.

[0077] Example 4: Anti-podoplanin monoclonal antibody targeting microbubbles

[0078] To demonstrate the versatility of the invention, this embodiment uses different targets and different clinical non-target controls.

[0079] The specific preparation method of the anti-podoplanin monoclonal antibody targeted microbubbles is the same as in Example 1, but the targeting part is replaced with anti-podoplanin monoclonal antibody (clone NZ-1, purchased from Wako). Definity (Lantheus Medical Imaging, USA; containing C3F8 microbubbles, a marketed non-targeted contrast agent) was used for clinical control.

[0080] In vitro binding assays were performed using human lung cancer cells A549 (highly expressing podoplanin) to construct tumor thrombosis mimics, following the same method as in Example 2. The results are shown in Table 6.

[0081] Table 6

[0082]

[0083] The above results indicate that the number of targeted microvesicles adhering to tumor thrombi is 7.8 times that of Definity (298 vs 38). The tumor / normal thrombus signal ratio is 7.1, which can be used for highly specific differentiation.

[0084] In vivo imaging experiments used the PANC-1 tumor thrombosis model (same as in Example 3). In Example 4, the peak signal enhancement of the targeted microbubbles was 15.2 ± 2.0 dB, and the Definity was 2.9 ± 0.5 dB (n=6, p<0.001).

[0085] Example 5: Fibrin-targeting cyclic peptide microvesicles (a universal thrombosis treatment)

[0086] The cyclic peptide synthesis and coupling steps are as follows: The linear peptide Cys-Arg-Glu-Lys-Ala-Gln-Gln-Cys (CREKAQC, SEQ ID NO.1) was synthesized in a solid-phase environment. Under weakly alkaline conditions (0.1M NH4HCO3, pH 8.0, shaking at room temperature for 24 hours), disulfide bonds were formed through thiol oxidation. Purification was performed using C18 reversed-phase HPLC, and the molecular weight was determined by mass spectrometry (theoretical value: 942.5, measured value: 943.2 [M+H]). + ) Dissolve DSPE-PEG2000-Mal in DMSO, add cyclic peptide (molar ratio 1:1.2), react at room temperature for 4 hours, purify and lyophilize.

[0087] The microbubble preparation method was the same as in Example 1, but DSPE-PEG-cyclic peptide was used instead of DSPE-PEG-antibody. The thrombosis binding assay results are shown in Table 7.

[0088] Table 7

[0089]

[0090] Example 5: The number of adhesions of targeted microbubbles to tumor thrombi and ordinary thrombi was not significantly different (p>0.05), and the two types of thrombi could not be distinguished. However, the absolute number of adhesions (236) was 6.9 times that of SonoVue (34), which is suitable for the general detection of thrombi (without distinguishing tumor-related factors).

[0091] In practical applications, if the clinical need is to "detect thrombi (without distinguishing the type)," then Example 5 can be selected; if the clinical need is to "distinguish between tumor thrombi and ordinary thrombi," then Example 1 (TF-targeted) or Example 4 (PDPN-targeted) should be selected.

[0092] Example 6: Comparison of diagnostic efficacy for different targets

[0093] The experimental data based on Examples 1-5 are summarized in Table 8.

[0094] Table 8

[0095]

[0096] Conclusion: For the clinical need of "diagnosing tumor thrombosis" (differentiating tumor thrombosis from ordinary thrombosis), TF and PDPN targeted microbubbles are significantly superior to fibrin targeted microbubbles.

[0097] Example 7: Stability of lyophilized formulations

[0098] The targeted microbubbles of Example 1 were freeze-dried and stored at 4°C. They were then reconstituted and tested after 0, 1, 3, and 6 months, respectively. The experimental results are shown in Table 9.

[0099] Table 9

[0100]

[0101] In summary, the lyophilized formulation maintains good stability within 6 months, meeting the requirements for clinical logistics and use.

[0102] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An ultrasound contrast agent targeting tumor thrombosis, characterized in that, include: The microbubble contains a perfluorinated carbon gas core, a microbubble shell covering the microbubble core and composed of an amphiphilic film-forming material, and a targeting portion specifically binding to tumor thrombosis-related markers attached to the outer surface of the shell; wherein the tumor thrombosis-related markers are selected from tissue factor, podoplanin, fibrin, activated platelet membrane glycoprotein GPIIb / IIIa, or P-selectin.

2. The ultrasound contrast agent targeting tumor thrombosis according to claim 1, characterized in that, The targeted portion is covalently connected to the microbubble shell layer via a DSPE-PEG connecting arm.

3. The ultrasound contrast agent targeting tumor thrombosis according to claim 1, characterized in that, The perfluorocarbon gas is selected from SF6, C3F8, or C4F. 10 .

4. The ultrasound contrast agent targeting tumor thrombosis according to claim 1, characterized in that, The average diameter of the microbubbles is 1-5 μm.

5. A method for preparing the ultrasound contrast agent targeting tumor thrombosis according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Couple the film-forming material with the target portion to obtain a target ligand-film-forming material conjugate; (2) The coupling material from step (1) is mixed with additional film-forming material in proportion, dissolved in an organic solvent, and rotary evaporated to form a thin film; (3) Add buffer solution to hydrate the film, and prepare microbubbles by mechanical vibration under a perfluorinated carbon gas atmosphere; (4) Purify to obtain targeted microbubbles.

6. The use of the ultrasound contrast agent targeting tumor thrombosis as described in any one of claims 1-4 in the preparation of reagents for ultrasound imaging diagnosis of tumor thrombosis.

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