A dual-response phase-change molecular probe for releasing disulfiram, and its preparation method and application

Abstract

The present invention discloses a dual-responsive phase-change molecular probe for releasing disulfiram, and its preparation method and application, and specifically relates to the field of biomedicine technology. The present invention first performs surface modification on the MMP-2-responsive PEGylated peptide EGPLGVRGK, and obtains a PEGylated MMP-2 peptide segment by covalent coupling; then the PEGylated MMP-2 peptide segment is covalently coupled to the surface of the PLGA-COOH shell layer, and a standard coupling agent method is used to form a PLGA-MMP2-PEG complex; finally, the PLGA-MMP2-PEG complex is reacted with DSF and PFP to obtain PFP@PDM-PEG nanoparticles. The dual-responsive phase-change molecular probe for releasing disulfiram of the present invention paves the way for effective cancer treatment strategies by using LIFU irradiation to destroy the complex microvascular environment in the tumor; it has intelligent design and biocompatible functionalization, and can inhibit tumor growth and metastasis by resisting angiogenic mimicry (VM), providing a strategic and innovative method for cancer treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a dual-response disulfiram-releasing phase-change molecular probe, a preparation method thereof, and an application thereof. Background Art

[0002] Currently, many cancers, especially triple-negative breast cancer (TNBC), are poorly treated due to their complex microvascular environment.

[0003] Breast cancer is a highly vascular-dependent tumor, and angiogenesis is a key process in its growth and metastasis. Therefore, inhibiting breast cancer angiogenesis can block the blood supply and nutrients to the tumor, thereby inhibiting its growth and metastasis. Currently, drugs targeting breast cancer angiogenesis mainly include anti-vascular endothelial growth factor (VEGF) antibodies and angiostatic factor (VEGFR) kinase inhibitors. These drugs inhibit the VEGF signaling pathway through different mechanisms, thereby inhibiting angiogenesis. However, the clinical application of anti-angiogenic therapy in breast cancer has been limited. Although anti-angiogenic therapy has achieved some success in non-small cell lung cancer, renal cell carcinoma, and hepatocellular carcinoma, its value in breast cancer is considered relatively limited. On the one hand, breast cancer has a low dependence on endothelial cell-dependent angiogenesis, so inhibiting angiogenesis has limited therapeutic efficacy in breast cancer. On the other hand, after chemotherapy or anti-angiogenic therapy, breast cancer lesions undergo adaptive changes in endothelial cell-dependent angiogenesis, which may lead to reversal of treatment efficacy and worsening prognosis.

[0004] In breast cancer, abnormalities in the structure and function of newly formed tumor blood vessels lead to an imbalance between tumor growth and oxygen supply, creating a hypoxic and weakly acidic microenvironment. This microenvironment prompts tumor cells to produce large amounts of matrix metalloproteinase-2 (MMP-2), which damages the basement membrane and degrades the extracellular matrix, forming a network structure capable of transporting blood, known as vasculogenic mimicry (VM). VM participates in reshaping the tumor's vascular microenvironment, with the luminal area of VM being 11.6 times that of endothelial-dependent vessels. The presence of VM exacerbates the imbalance in interstitial pressure and oxygen permeability within the tumor and has been shown to be a hallmark of poor prognosis in breast cancer. Currently, the application of anti-angiogenic therapy in breast cancer is limited. Breast cancer has a low dependence on endothelial-dependent angiogenesis, so inhibiting angiogenesis has limited therapeutic efficacy. Furthermore, after chemotherapy or anti-angiogenic therapy, breast cancer undergoes adaptive changes within the tumor lesion, increasing the formation of VM and further exacerbating the tumor's hypoxic state.

[0005] Tumor vasculogenic mimicry (VM) is a key structure in TNBC that can influence drug delivery and distribution. Although many drug delivery systems have been developed, targeted drug delivery to VM-dense tumor regions remains a challenge. Therefore, developing a novel drug delivery system that can effectively accumulate drugs in VM-dense areas is key to addressing this issue.

[0006] Studies have found that disulfiram (DSF) is a potential inhibitor of VM development. DSF has been used to treat alcoholism for decades and has shown anti-tumor effects. It has the ability to inhibit the activity of matrix metalloproteinase 2, which can inhibit tumor metastasis and reduce the growth activity of tumors that are defective in existing technology solutions. Therefore, DSF may become a new drug in the anti-angiogenic treatment of breast cancer, with high economic and social benefits. However, the application of DSF also has some problems, such as insolubility in water and short half-life. Therefore, the development of molecular probes that integrate imaging and treatment carrying DSF is a very promising strategy for the treatment of breast cancer. Summary of the Invention

[0007] To this end, the present invention provides a dual-responsive disulfiram-releasing phase-change molecular probe and a preparation method and application thereof, in order to solve the problem of poor therapeutic effects of existing cancers, especially breast cancer.

[0008] The molecular probe of the present invention is an integrated molecular probe for imaging and treatment of DSF. At the same time, the molecular probe can be efficiently targeted and accumulated in the tumor microvascular environment under the guidance of low-focus ultrasound imaging, and can achieve effective controlled release of DSF.

[0009] The molecular probe of the present invention is used to enhance drug accumulation in the central area of the tumor, thereby inhibiting tumor angiogenic mimicry (VM); utilizing the MMP-2 responsive drug delivery mechanism and surface modification using the MMP-2 responsive PEGylated peptide EGPLGVRGK-PEG to achieve targeted drug delivery and cellular uptake; in addition, imaging-guided low-intensity focused ultrasound (LIFU) is integrated to precisely control drug release and further inhibit VM and tumor growth.

[0010] In order to achieve the above object, the present invention provides the following technical solutions:

[0011] According to the first aspect of the present invention, a method for preparing a dual-responsive phase-change molecular probe for releasing disulfiram comprises:

[0012] Step 1: Surface modification of the MMP-2-responsive PEGylated peptide EGPLGVRGK was performed to obtain a PEGylated MMP-2 peptide segment by covalent coupling;

[0013] Step 2: Covalently couple the PEGylated MMP-2 peptide to the PLGA-COOH shell surface using standard coupling reagents to form a PLGA-MMP2-PEG complex.

[0014] Step 3: The PLGA-MMP2-PEG complex reacts with DSF and PFP through ultrasonic emulsification to obtain PFP@PDM-PEG nanoparticles, which are dual-responsive phase change molecular probes that release disulfiram.

[0015] Furthermore, in the step 1, the MMP-2 responsive PEGylated peptide EGPLGVRGK has MMP-2 enzyme affinity.

[0016] Furthermore, in the step 2, the coupling agent is carbodiimide.

[0017] According to the second aspect of the present invention, a dual-responsive phase-change molecular probe for releasing disulfiram is provided. The molecular probe is a DSF-loaded nanosystem, which is surface-modified with the MMP-2-responsive PEGylated peptide EGPLGVRGK to achieve targeted drug delivery and cellular uptake.

[0018] According to the third aspect of the present invention, a dual-responsive phase-change molecular probe that releases disulfiram is used in the preparation of a drug for inhibiting the spread of triple-negative breast cancer, which effectively inhibits the progression and spread of triple-negative breast cancer (TNBC) by inhibiting VM.

[0019] According to the fourth aspect of the present invention, a dual-responsive phase-change molecular probe for releasing disulfiram is used in integrated imaging-guided low-intensity focused ultrasound, which can be used to precisely control the release of drugs and inhibit VM and tumor growth.

[0020] According to a fifth aspect of the present invention, a dual-responsive phase-change molecular probe for releasing disulfiram is provided for use in the preparation of anti-tumor drugs.

[0021] Furthermore, the anti-tumor mechanism is biocompatible functionalization, which inhibits tumor growth and metastasis by resisting angiogenic mimicry VM.

[0022] The present invention has the following advantages:

[0023] The dual-responsive phase-change molecular probe for releasing disulfiram of the present invention achieves targeted delivery and precisely controlled release of drugs by using an MMP-2 responsive drug delivery mechanism and imaging-guided low-intensity focused ultrasound (LIFU) response, thereby further inhibiting VM and tumor growth; at the same time, it can enhance the accumulation of drugs in the central area of the tumor, thereby effectively inhibiting tumor VM.

[0024] The dual-responsive disulfiram-releasing phase-change molecular probe of the present invention paves the way for effective cancer treatment strategies by using LIFU irradiation to destroy the complex microvascular environment in tumors; it has intelligent design and biocompatible functionalization, and can inhibit tumor growth and metastasis by resisting VM, providing a strategic and innovative approach for cancer treatment.

[0025] The present invention efficiently reuses the FDA-approved anti-alcohol drug DSF, enhancing the accumulation of DSF in tumor areas with dense angiogenic mimicry through MMP-2 responsive drug delivery; facilitates cellular uptake of the drug through dePEGylation of MMP-2; guides controlled drug release through low-intensity focused ultrasound imaging, promoting its deep penetration and uniform dispersion in the tumor; and inhibits cancer, especially the progression and metastasis of triple-negative breast cancer, by inhibiting angiogenic mimicry.

[0026] The present invention develops a simple, safe and effective anti-cancer method, especially a method for treating triple-negative breast cancer angiogenesis. This method effectively inhibits tumor recurrence and metastasis by inhibiting angiogenic mimicry, showing potential for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0028] The structures, proportions, sizes, etc. illustrated in this specification are intended only to complement the contents disclosed herein and to facilitate understanding and reading by persons familiar with the art. They are not intended to limit the conditions under which the present invention may be implemented and therefore have no substantive technical significance. Any structural modifications, changes in proportions, or adjustments in sizes, without affecting the efficacy and objectives of the present invention, shall still fall within the scope of the technical contents disclosed herein.

[0029] Figure 1 Characterization of PFP@PD and PFP@PDM-PEG provided in Experimental Example 1 of the present invention, including: (A) synthesis diagram and 1H NMR analysis of PFP@PDM-PEG; (B) SEM and TEM images of PFP@PD and PFP@PDM-PEG, scale bar: 500 nm; (C) particle size of PFP@PDM-PEG and PFP@PDM-PEG; (D) zeta potential distribution of PFP@PD and PFP@PDM-PEG;

[0030] Figure 2 This is a graph showing the binding rate of MMP-2-PEG and PLGA-COOH provided in Experimental Example 1 of the present invention;

[0031] Figure 3 PLGA-MMP2-PEG reduces VM formation under ultrasound imaging guidance provided in Experimental Example 2 of the present invention, wherein A-changes in PFP@PDM-PEG after LIFU irradiation; B-ultrasound and enhanced ultrasound images captured before and after each LIFU irradiation; C-ultrasound imaging, CEUS peak imaging, and CEUS average sound intensity simulation heat map of the tumor site captured by LIFU irradiation 24 hours after the animal model was injected with PFP@PD and PFP@PDM-PEG in the tail vein; D-quantitative analysis of ultrasound intensity at the tumor site 24 hours after the animal model was injected with PFP@PD and PFP@PDM-PEG in the tail vein; E-quantitative analysis of CEUS average sound intensity at the tumor site 24 hours after the animal model was injected with PFP@PD and PFP@PDM-PEG in the tail vein;

[0032] Figure 4 This is a diagram of the in vivo targeted VM treatment provided in Experimental Example 2 of the present invention; wherein, A-comparison of the VM structure (red circle) and the number of endothelial cell-dependent blood vessels (brown circle) in tumor sections of each treatment group; B-fluorescence staining of COL1 and activated MMP-2 in tumor sections of each treatment group; C-protein immunoblot (Western Blot, WB) analysis of COL1 and activated MMP-2 in tumor sections of each treatment group; D-quantitative analysis of VM structure in tumor sections of each treatment group; E-quantitative analysis of endothelial cell-dependent blood vessels in tumor sections of each treatment group. DETAILED DESCRIPTION

[0033] The following describes the implementation of the present invention using specific embodiments. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. Obviously, the embodiments described are only a portion of the present invention, not all of it. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0034] The encapsulation efficiency (EE) and loading capacity (LC) of DSF were determined using a reverse phase (RP) high performance liquid chromatography system (1260 Infinity II, USA).

[0035] EE and LC were calculated according to the following formula (n=3):

[0036] EE = (weight of DSF in nanoparticles) / (total weight of added DSF) × 100%;

[0037] LC = (weight of DSF in nanoparticles) / (weight of nanoparticles) × 100%;

[0038] Ultrasound imaging-guided PLGA-MMP2-PEG reduces VM formation and its mechanism.

[0039] Example 1

[0040] 1. Surface modification with peptides and PEG:

[0041] 1.1 Peptide selection and design: The MMP-2 peptide (EGPLGVRGK) was selected and designed based on literature and has affinity for the MMP-2 enzyme.

[0042] 1.2 Peptide PEGylation Modification Methods:

[0043] PEG3000-COOH was used to introduce PEG groups, and the peptide PEG modification was achieved through covalent coupling with the MMP-2 peptide.

[0044] (1) First, the MMP-2 peptide (EGPLGVRGK) was synthesized using the Fmoc solid-phase peptide synthesis (SPPS) method, which has been reported.

[0045] (2) PEG3000-COOH was then introduced and washed sequentially with dimethylformamide (DMF), dichloromethane (DCM) and methanol in a reaction vessel and then dried.

[0046] (3) The mixture was then cleaved using a mixture of trifluoroacetic acid (TFA), water, 1,2-ethyldithiooctanoic acid (EDT), and triisopropylsilane (TIS), followed by sequential evaporation to remove the organic solvent DCM.

[0047] (4) Finally, the crude product was purified by dialysis against distilled water (MWCO 3,500 Da) to obtain the PEGylated MMP-2 peptide fragment.

[0048] 2. Preparation of PLGA-MMP2-PEG Complex

[0049] The PEGylated MMP-2 peptide was covalently coupled to the PLGA-COOH shell surface using a standard coupling agent (classical carbodiimide) method to form a PLGA-MMP2-PEG complex.

[0050] (1) Prepare the solution of PLGA-COOH and PEGylated MMP-2 peptide under laboratory conditions, ensuring that the operating table and laboratory equipment are clean and sterile.

[0051] (2) Place the PLGA-COOH solution and the peptide solution in two reaction tubes respectively, and prepare the carbodiimide activators: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

[0052] (3) In another small reaction tube, EDC and NHS are mixed in a certain molar ratio and dissolved in a solvent.

[0053] (4) Quickly add the activator solution to the PLGA-COOH solution and mix thoroughly. Slowly add the PEGylated MMP-2 peptide solution to the newly mixed reaction solution and gently shake or rotate the reaction tube to mix thoroughly overnight.

[0054] (5) After the reaction is completed, trifluoroacetic acid is added.

[0055] (6) Centrifugation and washing: The reaction mixture is centrifuged to separate the solid precipitate (i.e., the covalently coupled product) from the solution. The solid precipitate is washed with ether to remove unreacted substances and by-products.

[0056] (7) Purification: The washed product is dialyzed to remove impurities and solvents, and the PLGA-MMP2-PEG complex is freeze-dried for further use.

[0057] 3. Synthesis of PFP@PDM-PEG Nanoparticles

[0058] (1) A mixture of PLGA-MMP2-PEG complex (60 mg), DSF (4.8 mg) and PFP (200 μl) was dissolved in dimethyl sulfoxide (DMSO) and sonicated for 3 min (100 W, on:off = 1:1) using an ultrasonic processor (Sonics & Materials Inc., Newtown, CT, USA) under ice bath conditions.

[0059] (2) 8 ml of polyvinyl alcohol solution (4% w / v) was added and ultrasonic treatment was performed again under the same conditions.

[0060] (3) Isopropyl alcohol solution (10 ml, 2% v / v) was added, and the mixture was magnetically stirred in an ice bath for 3 hours to remove the organic solvent.

[0061] (4) After washing and suspension by centrifugation (4°C, 12,000 rpm, 8 min), the dual-responsive phase-change molecular probe PFP@PDM-PEG nanoparticles that release disulfiram were obtained.

[0062] Encapsulation efficiency (EE) is 75.7%;

[0063] The loading capacity (LC) is 6.8%. The encapsulation efficiency and loading capacity of this embodiment are the best, and the subsequent experiments are all carried out using the PFP@PDM-PEG nanoparticles of this embodiment.

[0064] Example 2

[0065] This example provides a method for preparing PLGA-MMP2-PEG, except that the DSF / PLGA-MMP2-PEG (weight ratio) is 1:2.5, and the rest is exactly the same as in Example 1.

[0066] Encapsulation efficiency (EE) is 8.3%;

[0067] The load capacity (LC) is 2.3%.

[0068] Example 3

[0069] This example provides a method for preparing PLGA-MMP2-PEG, except that the DSF / PLGA-MMP2-PEG (weight ratio) is 1:7.5, and the rest is exactly the same as in Example 1.

[0070] Encapsulation efficiency (EE) is 41.2%;

[0071] The load capacity (LC) is 5.2%.

[0072] Example 4

[0073] This example provides a method for preparing PLGA-MMP2-PEG, except that the DSF / PLGA-MMP2-PEG (weight ratio) is 1:17.5, and the rest is exactly the same as in Example 1.

[0074] Encapsulation efficiency (EE) is 70.5%;

[0075] The load capacity (LC) is 3.7%.

[0076] Example 5

[0077] This example provides a method for preparing PLGA-MMP2-PEG, except that the DSF / PLGA-MMP2-PEG (weight ratio) is 1:20, and the rest is exactly the same as in Example 1.

[0078] Encapsulation efficiency (EE) is 73.3%;

[0079] The load capacity (LC) is 3.4%.

[0080] Comparative Example

[0081] This comparative example provides a synthesis of PFP@PD nanoparticles: a mixture containing PLGA-COOH (50 mg), DSF (5 mg) and PFP (200 μl) was dissolved in DMSO and subjected to ultrasonic treatment. The subsequent steps were the same as the above process.

[0082] Experimental Example 1

[0083] 1. Nuclear magnetic resonance spectroscopy ( 1 H NMR) were recorded on an NMR spectrometer (BrukerAVANCE NEO400, BrukerBiospin GmbH, Rheinstetten, Germany) to confirm the successful covalent bonding between PLGA-COOH, MMP-2 substrate peptide and PEG. Figure 1 As shown in A.

[0084] 2. The morphology of the PFP@PDM-PEG nanoparticles of Example 1 and the comparative example PFP@PD nanoparticles was examined by transmission electron microscopy (TEM, Hitachi H-7500, Tokyo, Japan) and scanning electron microscopy (SEM, Hitachi SU8010, Tokyo, Japan). Figure 1 As shown in Figure B, both PFP@PDM-PEG and PFP@PD nanoparticles are evenly distributed spherical.

[0085] 3. Particle size and zeta potential were measured using a Malvern Zetasizer Nano ZS90 (UK). Figure 1 As shown in Figures C and 1D, the diameters of PFP@PDM-PEG and PFP@PD nanoparticles are approximately 343 nm and 305 nm, respectively, and the potentials are approximately -9.3 mV and -5.6 mV, respectively.

[0086] 4. To quantitatively evaluate the binding rate of FITC-labeled MMP-2-PEG to PLGA-COOH, flow cytometry (FCM) (Sonic SH800, Japan) was used, using PFP@PD nanoparticles as a control. The excitation wavelength was set at 488 nm, and the binding rate of MMP-2-PEG to PLGA-COOH reached 99%.

[0087] Experimental Example 2

[0088] Ultrasound imaging-guided PLGA-MMP2-PEG reduces VM formation and its mechanism

[0089] 1. In vitro phase transition confirmation

[0090] To confirm the acoustically induced phase transition capability of PFP@PDM-PEG nanoparticles, a low-intensity focused ultrasound device (LIFU, LMSC051 ACA; Institute of Ultrasound Imaging, Chongqing Medical University, Chongqing, China) was used to conduct experiments. The nanoparticles were excited by LIFU, and their phase transition process was observed under an optical microscope. After LIFU irradiation (2 W cm -2 , 3 minutes), the size of PFP@PDM-PEG increased from nanoscale to microscale. As the excitation time increased, the amount of phase-changed PFP@PDM-PEG increased ( Figure 3 A).

[0091] 2. In vitro ultrasound imaging (USI) performance: The in vitro ultrasound imaging (USI) performance of PFP@PDM-PEG nanoparticles was evaluated by diluting and placing them in a porous 3% agarose gel model. The samples were then exposed to pulsed LIFU radiation for 1 to 4 minutes at power levels of 1-4 W cm -2 Ultrasound images were captured before and after each LIFU exposure to determine the most appropriate LIFU parameters for in vivo evaluation. Figure 3 As shown in B, in LIFU (2W cm -2 ) for 3 minutes, the optimal ultrasound imaging effect of PFP@PDM-PEG was achieved. Quantitative analysis of echo intensity supported this finding, reaching a peak under these conditions, consistent with CEUS images.

[0092] 3. In vivo studies

[0093] Three mice bearing 4T1 tumors were randomly divided into two groups. Each group received PFP@PD nanoparticles and PFP@PDM-PEG nanoparticles (200 μL, PLGA concentration was equal to 1.0 mg mL -1 ) was injected intravenously. 24 hours later, US mode, CEUS mode (enhanced ultrasound) mode, and PI (peak intensity) mode images were acquired at the tumor site. -2 The ultrasound intensity values were analyzed using the DFY ultrasound imaging analysis software developed by the Institute of Ultrasound Imaging of Chongqing Medical University. Figure 3 As shown in C, 3D, and 3E.

[0094] 4. In vivo Targeted VM Therapy

[0095] To evaluate the antitumor effect of PFP@PDM-PEG nanoparticles in inhibiting VM formation in vivo, 4T1 tumor-bearing mice were randomly divided into different groups (n=5):

[0096] (i) Control group (ii) LIFU group (only LIFU irradiation) (iii) DSF group (iv) PFP@PD group (v) PFP@PDM-PEG group (vi) PFP@PDM-PEG + LIFU group. Mice were injected via the tail vein with 200 μL of saline, PFP@PD nanoparticles, PFP@PDM-PEG nanoparticles, or DSF suspension (equivalent DSF concentration: 10 mg kg -1 24 hours after injection, the tumor area was irradiated with LIFU (2 W cm -2 Tumor size was measured every two days, and tumor volume was calculated using the formula: (length × width^2) / 2 mm 3 . Two weeks later, blood samples were collected from the orbits and routine blood and biochemical indices were evaluated. Subsequently, each group of mice was euthanized, and H&E staining was performed on the main organs (heart, liver, spleen, lungs, and kidneys) to evaluate tissue pathological toxicity. Tumor tissues were examined using CD34 / PAS (Abcam, ab8536, Cambridge, Massachusetts), COL1 (affinitybiosciences, AF7001, Jiangsu, China), or activated MMP-2 (Abcam, ab92536, Cambridge, Massachusetts) staining to determine the mechanism by which PFP@PDM-PEG inhibits VM development, and to observe whether it affects endothelial cell-dependent tumor vasculature ( Figure 4 In addition, the protein levels of COL1 and activated MMP-2 in tumor tissues were quantified by Western blot analysis ( Figure 4 C), and quantitatively analyzed VM structure by comparing between groups ( Figure 4 D) and endothelial cell-dependent tumor vasculature ( Figure 4 E) average density.

[0097] PFP@PDM-PEG combined with low-focused ultrasound irradiation was able to maximize the inhibition of VM formation. This group also had the lowest levels of COL1 and activated MMP-2 proteins, indicating that the nanoparticles can reduce VM formation by downregulating COL1 and activated MMP-2 proteins. There were no differences in endothelial cell-dependent tumor vessels between treatment groups, indicating that the nanoparticles had no effect on them.

[0098] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for preparing a dual-responsive phase-change molecular probe for releasing disulfiram, characterized in that: include: Step 1: Surface modification of the MMP-2 responsive peptide EGPLGVRGK was performed to obtain a PEGylated MMP-2 peptide segment by covalent coupling; Step 2: Covalently couple the PEGylated MMP-2 peptide to the PLGA-COOH shell surface using standard coupling reagents to form a PLGA-MMP2-PEG complex. Step three, the PLGA-MMP2-PEG complex is subjected to ultrasonic emulsification reaction with DSF and PFP to obtain PFP@PDM-PEG nanoparticles, which are dual-responsive phase change molecular probes that release disulfiram.

2. The method for preparing a dual-responsive phase-change molecular probe for releasing disulfiram according to claim 1, characterized in that: In the step 1, the MMP-2 responsive peptide EGPLGVRGK has MMP-2 enzyme affinity.

3. The method for preparing a dual-responsive phase-change molecular probe for releasing disulfiram according to claim 1, characterized in that: In the step 2, the coupling agent is carbodiimide.

4. The dual-responsive phase-change molecular probe for releasing disulfiram prepared by the method according to any one of claims 1 to 3, characterized in that: The molecular probe is a nanosystem loaded with DSF.

5. Use of the dual-responsive disulfiram-releasing phase-change molecular probe according to claim 4 in the preparation of a drug for inhibiting the spread of triple-negative breast cancer.

6. Use of the dual-responsive disulfiram-releasing phase-change molecular probe according to claim 4 in integrated imaging-guided low-intensity focused ultrasound.

7. Use of the dual-responsive disulfiram-releasing phase-change molecular probe according to claim 4 in the preparation of anti-tumor drugs, characterized in that: The tumor is breast cancer.

8. The use according to claim 7, characterized in that The anti-tumor mechanism is to inhibit tumor growth and metastasis by resisting vasculogenic mimicry (VM).

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