MMP-2 activated type electric field enhanced acoustic afterglow nano probe and preparation method and application thereof

Abstract

The application discloses an MMP-2 activation type electric field enhanced acoustic afterglow nano probe and a preparation method and application thereof. The electric-acoustic afterglow nano probe is composed of an acoustic afterglow organic polymer light-emitting component, a piezoelectric component, an amphiphilic polymer and a responsive recognition unit containing an MMP-2 cleavable peptide segment and a quenching group. In the unactivated state, the quenching group inhibits the light signal of the light-emitting component, so that the probe maintains a low background; in the tumor microenvironment with high activity of MMP-2, the responsive peptide segment is specifically cleaved, the quenching group is separated from the light-emitting component, and thus the electric-acoustic afterglow light-emitting is restored. The nano probe has the advantages of enzyme response low background and electric field enhanced acoustic afterglow light-emitting, and solves the problems that the existing MMP-2 detection methods are difficult to realize in-situ spatial positioning in the body, the early lesion signal is weak, and the recognition sensitivity of the micro metastasis is insufficient.

Description

Technical Field

[0001] This invention belongs to the field of acoustic afterglow luminescence material research and application, specifically involving a matrix metalloproteinase-2 (MMP-2) activated electric field enhanced acoustic afterglow nanoprobe, its preparation method and application. Background Technology

[0002] 1. The Needs and Core Challenges of Imaging Early Tumor Lesions and Micrometastases Early tumor lesion imaging and micrometastasis identification are crucial for lesion localization, treatment planning, and recurrence risk assessment. However, early-stage tumors are typically small and lack significant anatomical and morphological changes, making it difficult for conventional imaging methods to achieve sufficient contrast. This is especially true for deep tumors and micrometastases in organs such as the lungs, where low signal intensity and complex tissue background further limit imaging sensitivity. Traditional imaging primarily relies on tumor volume or morphological changes, and its detection time is usually limited by lesion size, making it difficult to reflect early molecular events during tumor development and metastasis. Therefore, developing in vivo imaging techniques that can convert abnormal activation of tumor-related molecules into high-contrast imaging signals is an important way to improve the identification of early lesions and micrometastases.

[0003] 2. Advantages of MMP-2 activity imaging and limitations of existing detection technologies Matrix metalloproteinase-2 (MMP-2) is primarily involved in extracellular matrix degradation, basement membrane disruption, and tumor invasion front remodeling. Its activation level typically increases before obvious changes in tumor anatomy. Compared to imaging methods that rely on tumor volume or morphological changes, in situ detection of MMP-2 activity is more advantageous in reflecting abnormal molecular activity in the tumor microenvironment before significant anatomical changes occur, and can be used to visualize tumor invasion areas and micrometastases. Current MMP-2 detection methods mostly rely on in vitro tissue analysis or body fluid marker detection. While these methods can reflect certain changes in MMP-2 expression or content, they struggle to provide spatial location information of tumor lesions in vivo and to dynamically reflect the enzyme activity distribution at the tumor invasion front and in micrometastases. Some fluorescent probes can generate signals through MMP-2 cleavage response units, but these typically require continuous excitation from an external light source and are susceptible to the effects of tissue autofluorescence, excitation light scattering, and tissue absorption. Therefore, their imaging signal-to-noise ratio and detection sensitivity remain limited in deep, small lesions. Therefore, there is an urgent need to develop novel response probes suitable for in situ in vivo MMP-2 activity imaging.

[0004] 3. Technical requirements for MMP-2 activation and piezoelectric localized electric field-enhanced acoustic afterglow imaging Acoustic afterglow imaging utilizes ultrasound to deeply activate luminescent materials and acquires a continuous afterglow signal after ultrasound stops, separating the energy excitation process from the signal acquisition process, thereby reducing the interference of external excitation and tissue background on the imaging results. Introducing cleavable peptides and quenching groups of MMP-2 into the acoustic afterglow nanosystem allows the probe to maintain low background in its unactivated state; when the probe enters the highly active region of MMP-2, the responsive peptide is cleaved, and the quenching group separates from the luminescent component, converting MMP-2 enzyme activity into a restored acoustic afterglow signal. However, the probe enrichment and the number of MMP-2 response events in early tumors and micrometastases are relatively limited. Ordinary MMP-2-responsive acoustic afterglow probes may still suffer from insufficient luminescence intensity and low photon output efficiency after enzymatic activation, making it difficult to meet the high-sensitivity imaging requirements of deep micro-lesions. Piezoelectric materials can generate a piezoelectric effect by polarization under ultrasonic mechanical stress. By nanoscale composite of piezoelectric components with an MMP-2-responsive acoustic afterglow luminescence system, electro-acoustic afterglow luminescence can be synchronously achieved after MMP-2 response using the same ultrasonic input, thereby further amplifying the acoustic afterglow signal after MMP-2 enzymatic digestion. Therefore, it is necessary to construct an acoustic afterglow nanoprobe that combines MMP-2 specific response with afterglow signal enhancement to improve the sensitivity and signal-to-noise ratio of in vivo MMP-2 activity imaging and to use it for in situ identification of early tumor lesions and tumor invasion areas. Summary of the Invention

[0005] This invention provides an MMP-2-activated electric field-enhanced acoustic afterglow nanoprobe, its preparation method, and its applications. The nanoprobe is constructed from an organic polymer luminescent component with acoustic afterglow luminescence properties, a piezoelectric component, an MMP-2 responsive recognition unit, a quenching group, and surface modification materials. The responsive recognition unit includes a cleavable MMP-2 peptide. In the inactive state, the quenching group inhibits the light signal of the organic polymer luminescent component, maintaining a low background for the nanoprobe. When the nanoprobe enters a highly active MMP-2 region, the cleavable peptide is specifically cleaved, separating the quenching group from the organic polymer luminescent component, thereby restoring the afterglow luminescence signal. Under ultrasound, the piezoelectric component polarizes and generates a local electric field, further amplifying the afterglow signal after enzymatic cleavage by regulating the electronic structure and luminescence process of the organic polymer luminescent molecules. The acoustic afterglow signal of the nanoprobe increases with increasing MMP-2 activity or concentration, and can be used for in vivo in situ imaging of MMP-2 activity, particularly suitable for highly sensitive identification of early tumor lesions, tumor invasion areas, and micrometastases.

[0006] A MMP-2-activated electric field-enhanced acoustic afterglow nanoprobe comprises an organic polymer SP11 with acoustic afterglow luminescence properties; a piezoelectric component BTO capable of generating a piezoelectric response under ultrasonic mechanical stress; an amphiphilic polymer for coating and stabilizing the SP11 and BTO; an MMP-2 cleavable peptide and a quenching group IRQ; the SP11, BTO, and amphiphilic polymer are co-precipitated and self-assembled into composite nanoparticles, with the MMP-2 cleavable peptide and quenching group coupled to the surface; in the presence of MMP-2, the MMP-2 cleavable peptide is specifically cleaved, causing the quenching group to move away from the organic polymer luminescent component, thereby restoring the acoustic afterglow luminescence signal; under ultrasonic action, the piezoelectric component generates a local electric field and further amplifies the restored acoustic afterglow signal.

[0007] A method for preparing MMP-2-activated electric field-enhanced acoustic afterglow nanoprobes includes the following steps: dissolving or dispersing an organic polymer SP11 with acoustic afterglow luminescence properties, a piezoelectric component BTO, and an amphiphilic polymer with maleimide groups in an organic solvent; adding the resulting organic phase to an aqueous mixed medium for self-assembly; and removing the organic solvent to obtain maleimide-modified electric field-enhanced acoustic afterglow nanoprobes SPBT-Mal-NPs; and then adding MMP-2 cleavable peptides with free thiol groups and free amino groups. The SPBT-Mal-NPs were mixed to allow the free thiol groups to undergo an addition reaction with the maleimide groups, and after purification, the MMP-2-responsive electric field-enhanced acoustic afterglow nanoprobe SPBT-MMP-NH2 was obtained. The SPBT-MMP-NH2 was then mixed with an NHS-activated IRQ quencher to allow the IRQ quencher to undergo an amidation reaction with the free amino groups on the cleavable peptide of MMP-2, and after purification, the MMP-2-activated electric field-enhanced acoustic afterglow nanoprobe SPBT-MMP-IRQ was obtained.

[0008] Furthermore, the organic polymer SP11 with acoustic afterglow luminescence properties of the present invention has the following structure: ; The preparation method is as follows: Under a nitrogen protective atmosphere, tin compounds are added in a set proportion. The mixture of bromide (Br-A-Br) and tetra(triphenylphosphine)palladium was heated to 100-180 °C and stirred for 12-48 hours. After the reaction was completed, the mixture was filtered, purified, and dried to obtain the target product, compound SP11, with a degree of polymerization n of 2-1000.

[0009] Furthermore, the amphiphilic polymer with maleimide groups of the present invention is DSPE-PEG2000-Mal, and the organic solvent is tetrahydrofuran.

[0010] Furthermore, the amounts of SP11, barium titanate, and DSPE-PEG2000-Mal in this invention are 100 μg, 200 μg, and 2.5 mg, respectively. The volume ratio of the organic phase to the aqueous phase in the mixed phase is 1:5 to 1:20, preferably 1:9, and the self-assembly treatment time is 10 to 30 min.

[0011] Furthermore, the MMP-2 cleavable peptide of the present invention is NH 2 -GPLGIAGC-SH, in the presence of tris(2-carboxyethyl)phosphine, its terminal thiol group undergoes an addition reaction with the maleimide group on the surface of SPBT-Mal-NPs to form a thioether bond, yielding SPBT-MMP-NH2 with free amino groups on the surface. Furthermore, the SPBT-MMP-NH2 of the present invention undergoes an amidation reaction with an NHS-activated IRQ quencher to obtain SPBT-MMP-IRQ.

[0012] Furthermore, the amidation reaction of the present invention is carried out in an aqueous organic mixed medium at a reaction temperature of 20-40 °C for a reaction time of 1-8 hours, wherein the concentration of NHS-IRQ in the reaction system is 0.05-0.30 mmol / L, and the reaction is purified by ultrafiltration after completion.

[0013] An application of a nanoprobe for identifying different stages of tumor lesions and micrometastases. The tumor lesions are selected from one or more subcutaneous breast cancer tumors and lung tumor metastases.

[0014] By adopting the above technical solution, the present invention has the following beneficial effects: 1. MMP-2 Specific Activation and Low Background Signal Maintenance. This invention introduces MMP-2-cleavable peptides and IRQ quenching groups onto the surface of an electro-acoustic afterglow nanoprobe. This allows the probe to remain in a quenched state when not in contact with MMP-2, reducing non-specific background signal. When MMP-2 specifically cleaves the responding peptide, the IRQ separates from the luminescent component, thereby restoring fluorescence and acoustic afterglow luminescence. The probe exhibits good MMP-2 selectivity and is not easily interfered with by other common biological enzymes and reducing substances.

[0015] 2. The piezoelectric localized electric field significantly amplifies the acoustic afterglow signal after enzyme digestion activation. Ultrasonic stimulation can simultaneously trigger acoustic afterglow luminescence and piezoelectric component polarization, generating a localized electric field within the nanoprobe. This localized electric field can modulate the electron distribution and excited-state decay process of the luminescent component, thereby further amplifying the weak afterglow signal after MMP-2 enzyme digestion and quenching. Compared to MMP-2-responsive acoustic afterglow probes without piezoelectric components, the nanoprobe described in this invention exhibits higher afterglow luminescence intensity and imaging signal-to-noise ratio.

[0016] 3. Improved detection sensitivity for early-stage tumors and micrometastases. The nanoprobe described in this invention can convert the increased MMP-2 activity during tumor development, invasion, and metastasis into a low-background, high-contrast acoustic afterglow imaging signal, which can be used for in vivo imaging of early-stage tumor lesions, invasive areas, and micrometastases in the lungs. Compared with conventional acoustic afterglow control probes, the nanoprobe can obtain detectable signals at an earlier stage of lesion formation and when the lesion volume is small, thereby improving the imaging sensitivity and identification of early-stage lesions and micrometastases. Attached Figure Description

[0017] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0018] Figure 1 This diagram illustrates the synthesis and application of the MMP-2-activated electric field-enhanced acoustic afterglow nanoprobe prepared according to the present invention. Figure 2 The chemical reaction formula of the acoustic afterglow organic polymer SP11 prepared in Example 1 of this invention; Figure 3 The hydrogen nuclear magnetic resonance spectrum of the acoustic afterglow organic polymer SP11 prepared in Example 1 of this invention; Figure 4 The UV and fluorescence spectra of the MMP-2 activated electric field enhanced acoustic afterglow nanoprobe (SPBT-MMP-IRQ) prepared in Example 2 of this invention are shown. Figure 5 The images shown are transmission electron microscope images and particle size distribution diagrams of SPBT-MMP-IRQ obtained in Example 2 of this invention. Figure 6 The voltage generation test diagram of SPBT-MMP-IRQ obtained in Embodiment 2 of the present invention under non-ultrasonic and ultrasonic conditions; Figure 7 The fluorescence response curves of SPBT-MMP-IRQ to different concentrations of MMP-2 in Example 3 of this invention are shown. Figure 8 The images show the fluorescence signal and fluorescence imaging of the SPBT-MMP-IRQ obtained in Example 3 of this invention after being processed under different conditions. Figure 9 This is an image showing the afterglow response of SPBT-MMP-IRQ to different concentrations of MMP-2 in Example 3 of the present invention; Figure 10 These are afterglow images of SPBT-MMP-IRQ and control materials after being processed under different conditions in Example 3 of this invention; Figure 11 This is a afterglow imaging image of the selective response of SPBT-MMP-IRQ to different biological enzymes and reducing substances in Example 3 of the present invention. Figure 12 This is a cell viability graph measured by the CCK-8 assay using the nanoprobe in Example 3 of this invention; Figure 13 This is an in vivo acoustic afterglow luminescence imaging image of the SPBT-MMP-IRQ and control probes at different stages of development of subcutaneous tumors in mice, as shown in Example 4 of this invention. Figure 14 This is an in vivo acoustic afterglow luminescence imaging image of the SPBT-MMP-IRQ and control probes at different stages of the formation of micrometastatic lesions in the lungs of mice, as shown in Example 5 of the present invention. Detailed Implementation

[0019] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] This invention provides a method for preparing an MMP-2 activated electric field-enhanced acoustic afterglow nanoprobe, comprising: 1. The synthesis of an acoustic afterglow organic polymer molecule (SP11) with ultrasonically activated afterglow luminescence properties is provided. Specific synthetic reaction formulas can be found in the appendix of the instruction manual. Figure 2 The synthesis steps are as follows: Tin compound Sn-1 (218 mg, 0.21 mmol) and bromide Br-1 (143 mg, 0.21 mmol) were added to a three-necked flask and dissolved in 10 mL of toluene. The reaction system was evacuated and purged with nitrogen three times to establish a nitrogen-protected environment. Then, tetrakis(triphenylphosphine)palladium (9 mg, 0.01 mmol) was added, and the reaction system was heated to 120 °C and stirred for 24 hours. After the reaction was complete, the reaction solution was cooled to room temperature, and 200 mL of methanol was slowly added to allow the product to precipitate completely. The solid was collected by filtration and placed in a Soxhlet extractor. Extraction was performed sequentially with methanol, n-hexane, acetone, and chloroform, each solvent for 12 hours to remove unreacted monomers, oligomers, and other small molecule impurities. The chloroform extract was collected, concentrated by rotary evaporation, and then added dropwise to methanol to redefine the target product. Finally, after filtration, washing, and drying, the persistent organic polymer molecule SP11 was obtained, with a degree of polymerization n ranging from 2 to 1000.

[0021] 2. The synthesis of electro-acoustic afterglow nanoprobes (SPBT-Mal-NPs) based on the organic polymer molecule SP11 and piezoelectric components is provided. The synthesis steps are as follows: The organic phase was prepared by dissolving or uniformly dispersing the acoustic afterglow organic polymer SP11 (100 μg), the piezoelectric component barium titanate (BaTiO3, BTO, 100 μg), and distearate phosphatidylethanolamine-polyethylene glycol 2000-maleimide (DSPE-PEG2000-Mal, 2.5 mg) in 1 mL of tetrahydrofuran (THF). A sample without BTO was used as a control. The organic phase was rapidly added to a mixture of 9 mL of THF and deionized water (THF:water volume ratio 1:9) under ice bath, stirring, high-speed shearing, or ultrasonic conditions, and the mixture was stirred or ultrasonically treated for 10–30 minutes to allow the components to self-assemble into uniformly sized nanoparticles. The resulting dispersion was then placed in a shaker at 37 °C for 12 hours or subjected to rotary evaporation to further remove residual organic solvents and improve the stability of the nanoparticles. Finally, the dispersion was concentrated by centrifugation and ultrafiltration at 2000 rpm for 10 minutes, and the resulting nanoprobes were redispersed in ultrapure water and stored at 4 ℃ in the dark. The BTO-containing electro-acoustic afterglow nanoprobes were designated as SPBT-Mal-NPs; the BTO-free control acoustic afterglow nanoprobes were designated as SP-Mal-NPs.

[0022] 3. Preparation of MMP-2-activated electric field-enhanced acoustic afterglow nanoprobe (SPBT-MMP-IRQ). A stepwise chemical coupling method was used, specifically including two steps: coupling with MMP-2-responsive peptides and coupling with quenching groups. The specific synthesis steps are as follows: Weigh out 6.92 mg of MMP-2 cleavable peptides containing terminal thiol groups. 0.89 mg of tris(2-carboxyethyl)phosphine was dissolved in 10 mL of a mixed solvent of dimethyl sulfoxide (DMSO) and water at a volume ratio of 1:4. The solution was stirred at 37 °C for 60 min to reduce the terminal thiol groups of the peptides that might form disulfide bonds and keep them in a free state, yielding an MMP-2 responsive peptide solution. SPBT-Mal-NPs were prepared into an aqueous dispersion with a mass concentration of 0.1 mg / mL. 1 mL of this dispersion was added to the above peptide solution, and the mixture was stirred at room temperature in the dark for 5 h. The MMP-2 cleavable peptides were attached to the nanoprobe surface via a Michael addition reaction between the terminal thiol groups of the peptides and the maleimide groups on the nanoprobe surface. After the reaction, centrifugation and ultrafiltration were performed. The mixture was repeatedly washed and ultrafiltered with a mixed solvent of DMSO and water to remove unreacted MMP-2 responsive peptides, TCEP, and other small molecules. The sample was then washed further with deionized water or phosphate buffer to obtain SPBT-MMP-NH2 with free amino groups on its surface. The control group was SP-MMP-NH2. The obtained sample was prepared into a dispersion with an SP11 concentration of 30 μg / mL using ultrapure water, and 1 mL was used for later use. An NHS-IRQ solution was prepared using a 1:4 volume ratio of DMSO and water to achieve a final NHS-IRQ concentration of 0.15 mmol / L. The SPBT-MMP-NH2 dispersion was added to the NHS-IRQ solution, and the mixture was stirred at room temperature in the dark for 5 hours. This allowed the NHS-activated ester groups in NHS-IRQ to undergo amidation with the free amino groups at the end of the MMP-2 responsive peptide, thereby attaching the IRQ quencher groups to the end of the MMP-2 responsive peptide. After the reaction, the sample was subjected to centrifugation and ultrafiltration, and repeatedly washed with a 1:4 volume ratio of DMSO and water to remove unreacted NHS-IRQ and other small molecule impurities. The nanoprobes were then further washed with deionized water or phosphate buffer to obtain the MMP-2 activated electric field enhanced acoustic afterglow nanoprobe SPBT-MMP-IRQ (the control probe was SP-MMP-IRQ). The obtained nanoprobes were stored in the dark at 4 °C.

[0023] The MMP-2-activated electric field-enhanced acoustic afterglow nanoprobe prepared in this invention exhibits good water dispersibility and biocompatibility. In the unactivated state, the quencher group suppresses the fluorescence and acoustic afterglow signals of the organic polymer luminescent component through resonant energy transfer. When MMP-2 specifically cleaves the responsive peptide, the quencher group separates from the luminescent component, restoring the fluorescence and acoustic afterglow signals. In vitro experimental results show that the luminescence signal of the nanoprobe increases with increasing MMP-2 levels, and the acoustic afterglow luminescence can be further enhanced by utilizing the local electric field generated by the piezoelectric component under ultrasound. In vivo animal imaging results show that the nanoprobe can convert MMP-2 activity in tumor tissue and invasive metastatic lesions into a low-background, high-signal-to-noise ratio acoustic afterglow signal, which can be used for early tumor lesion imaging, tumor invasion area identification, and micrometastasis detection. This nanoprobe has good application potential in the fields of MMP-2 activity in situ imaging, early tumor detection, and tumor progression monitoring.

[0024] This invention characterizes the prepared acoustic afterglow organic polymer molecule SP11 using methods such as nuclear magnetic resonance (NMR); characterizes the prepared electro-acoustic nanoprobes using methods such as ultraviolet-visible-near-infrared absorption spectroscopy (UV-Vis), fluorescence spectrophotometer, transmission electron microscopy (TEM), dynamic light scattering analysis (DLS), and atomic force microscopy (AFM); evaluates the MMP-2 response performance and afterglow luminescence performance of the nanoprobes using a fluorescence spectrophotometer and IVIS spectral imaging system; evaluates the cytotoxicity of the nanoprobes using the CCK-8 assay; and finally tests the in vivo afterglow imaging performance of the nanoprobes in tumor-bearing mice. Specific test results are as follows: 1. NMR test results of organic polymer molecules with acoustic afterglow Reference manual attached Figure 3 The image shows the 1H NMR spectrum of the acoustic afterglow organic polymer SP11 prepared in this invention. SP11 exhibits characteristic proton signals corresponding to the expected molecular structure, and its chemical shift and peak shape are basically consistent with the target structure, indicating that the polymerization reaction proceeded smoothly and confirming that the acoustic afterglow organic polymer SP11 has been successfully prepared.

[0025] 2. UV-Vis and FL test results of the MMP-2 activated electric field enhanced acoustic afterglow nanoprobe SPBT-MMP-IRQ Reference manual attached Figure 4The UV-Vis-NIR absorption and fluorescence emission spectra of the unquenched nanoprobe SPBT-MMP-NH2, the quencher NHS-IRQ, and the quenched nanoprobe SPBT-MMP-IRQ are shown. The unquenched nanoprobe SPBT-MMP-NH2 exhibits the characteristic absorption of SP11 at approximately 608 nm and produces fluorescence emission in the 650–900 nm range; the quencher NHS-IRQ has a characteristic absorption at approximately 764 nm. SPBT-MMP-IRQ retains the characteristic absorption of SP11 at approximately 608 nm and also shows the characteristic absorption peak of IRQ at approximately 764 nm. Simultaneously, its fluorescence emission signal is significantly lower than that of SPBT-MMP-NH2, indicating that the IRQ quenching group can effectively suppress the fluorescence signal of the luminescent component, confirming the successful preparation of the quenched nanoprobe SPBT-MMP-IRQ. Furthermore, calculations show that each SPBT-MMP-IRQ can be linked to 150 IRQ quenching molecules.

[0026] 3. Results of transmission electron microscopy and hydration dynamics diameter measurement Reference manual attached Figure 5 The quenching nanoprobe SPBT-MMP-IRQ exhibits a spherical morphology with uniform size and good dispersion; the hydrodynamic particle size of the prepared quenching nanoprobe SPBT-MMP-IRQ is approximately 79.9 nm.

[0027] 4. Piezoelectric performance test results Reference manual attached Figure 6 Without the application of ultrasound, no obvious voltage signal was detected in the SPBT-MMP-IRQ; after the application of ultrasound, the SPBT-MMP-IRQ generated a voltage of about 50-100 mV, indicating that the piezoelectric component can be polarized and generate a piezoelectric potential under the action of ultrasonic mechanical stress.

[0028] 5. Study on the properties of fluorescence signals of nanoprobes activated by different concentrations of MMP-2 Reference manual attached Figure 7 Two mL of SPBT-MMP-IRQ at a concentration of 30 μg / mL based on SP11 were co-incubated with MMP-2 at concentrations of 0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 ng / mL, respectively. As the concentration of MMP-2 increased, the fluorescence signal of SPBT-MMP-IRQ gradually recovered and enhanced, exhibiting a clear concentration-dependent effect. This indicates that MMP-2 can specifically cleave the responsive peptide on the probe surface, separating the IRQ quenching group from the SP11 luminescent component, thereby dequenching and restoring fluorescence emission.

[0029] 6. Results of activated nanoprobe fluorescence signals after treatment under different conditions Reference manual attached Figure 8 Under simple sonication conditions, SPBT-MMP-IRQ produced almost no fluorescence signal, indicating that the IRQ quencher group can effectively suppress the fluorescence emission of SP11, allowing the nanoprobe to maintain a low background signal. After adding 200 µL of MMP-2 at a concentration of 100 ng / mL, the fluorescence signal of the nanoprobe was significantly restored, indicating that MMP-2 can specifically cleave the responsive peptide, separating the IRQ quencher group from the SP11 luminescent component, thereby relieving the quenching effect caused by resonance energy transfer and restoring fluorescence emission. After adding MMP-2 and applying sonication, the instantaneous fluorescence signal of the system was reduced compared to the group with only MMP-2 added, providing an optical possibility for the generation of afterglow signal.

[0030] 7. Study on the properties of afterglow luminescence signals of nanoprobes activated by different concentrations of MMP-2 Reference manual attached Figure 9 Two mL of SPBT-MMP-IRQ at a concentration of 30 μg / mL based on SP11 was co-incubated with MMP-2 at concentrations of 0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 ng / mL. As the MMP-2 concentration increased, the afterglow luminescence signal of SPBT-MMP-IRQ gradually recovered and strengthened, exhibiting a clear concentration dependence. This indicates that MMP-2 can specifically cleave the responsive peptide on the probe surface, separating the IRQ quenching group from the SP11 luminescent component, thereby dequenching and restoring afterglow luminescence.

[0031] 8. Afterglow luminescence results of nanoprobes treated under different conditions Reference manual attached Figure 10 Without the application of MMP-2, neither SPBT-MMP-IRQ nor SP-MMP-IRQ produced a significant afterglow signal, indicating that acoustic afterglow luminescence requires ultrasonic activation. When only ultrasound was applied without the addition of MMP-2, the ultrasonic power was 2.5 W / cm². 2 With an ultrasound duration of 60 seconds, neither probe showed a significant afterglow signal, indicating that the IRQ effectively maintained the quenched state, and ultrasound itself could not remove the quenching. After adding 200 µL of MMP-2 at a concentration of 100 ng / mL and applying ultrasound, SP-MMP-IRQ produced only a weak afterglow signal, while SPBT-MMP-IRQ containing the piezoelectric component produced a significantly enhanced afterglow signal, 50-200 times stronger than the control group. This indicates that MMP-2's specific cleavage of the responsive peptide can remove the quenching of the luminescent component by the IRQ and restore acoustic afterglow luminescence, while the localized electric field generated by the piezoelectric component further amplifies the afterglow signal activated by MMP-2.

[0032] 9. Research on anti-interference performance Reference manual attached Figure 11 The MMP-2 treatment group produced a significantly enhanced acoustic afterglow signal, while the PBS, GDH, Try, AChE, Caspase, NTR, NADH, ALP, and GranB treatment groups did not show significant signal enhancement. This indicates that SPBT-MMP-IRQ can selectively respond to MMP-2, its responsive peptide can be specifically recognized and cleaved by MMP-2, and it has good anti-interference ability against other common biological enzymes and reducing substances.

[0033] 10. Cytotoxicity results of nanoprobes: Reference manual attached Figure 12 When SP-MMP-IRQ and SPBT-MMP-IRQ nanoprobes were co-incubated with 4T1 cells at concentrations of 0, 5, 10, 20, 30, and 50 μg / mL for 24 hours, the cell viability was above 80%, indicating that SP-MMP-IRQ and SPBT-MMP-IRQ nanoprobes had almost no toxicity to cells.

[0034] 11. Study on the imaging performance of nanoprobes on early lesions in subcutaneous tumor models Reference manual attached Figure 13 This image shows the in vivo imaging results of the electro-acoustic afterglow nanoprobe described in this invention at different developmental stages of 4T1 subcutaneous tumors. On days 2, 4, 6, 8, and 10 post-tumor inoculation, SPBT-MMP-IRQ or the control probe SP-MMP-IRQ (without piezoelectric components) was injected via the tail vein at a volume of 0.2 mL and a concentration of 2 mg / mL. After the probe reached the predetermined enrichment time of 24 h, the tumor area was subjected to ultrasound excitation at a power of 2.5 W / cm² for 60 s, and afterglow images were acquired immediately after ultrasound discontinuation using an IVIS system. On days 2-4 post-tumor inoculation, both probes showed only weak background signals, indicating that the probes are less prone to non-specific activation when the tumor lesions are not yet obvious or MMP-2 activity is low. As the tumor grew, a significant acoustic afterglow signal was detected in the tumor area by day 6 in the SPBT-MMP-IRQ group; while the SP-MMP-IRQ without the piezoelectric component produced a weaker acoustic afterglow signal under the same time point and ultrasound conditions, and the lesion was not detected until day 10. The results indicate that SPBT-MMP-IRQ can respond to the increased MMP-2 activity during tumor development and further amplify the weak afterglow signal after enzyme activation through a piezoelectric local electric field, thereby improving the imaging sensitivity and contrast of early small lesions.

[0035] 12. Study on the lesion recognition performance of nanoprobes for micrometastatic tumors Reference manual attached Figure 14This document presents in vivo imaging results of the micrometastasis development stage of the lung using the nanoprobe described in this invention. A mouse model of lung metastases was established, and at weeks 1, 2, 3, 4, and 5 of the metastasis development process, SPBT-MMP-IRQ or the control probe SP-MMP-IRQ (without piezoelectric components) was injected via the tail vein at a concentration of 2 mg / mL and a volume of 200 μL, respectively. After the probe reached the predetermined enrichment time of 24 h, the lung region was subjected to ultrasound excitation at a power of 2.5 W / cm² for 60 s, and afterglow luminescence images were acquired after the ultrasound stopped. In normal lung tissue and in the early stages of metastasis, both probe groups showed only weak background signals. With the formation of lung metastatic lesions and the increase in MMP-2 activity, clear micro-pulmonary afterglow signals were detectable in the SPBT-MMP-IRQ group as early as week 3, while weak signals were not observed in the SP-MMP-IRQ group until week 5. The results showed that SPBT-MMP-IRQ can effectively amplify the weak enzyme response signal in micrometastases through the synergistic effect of MMP-2 specific activation and piezoelectric local electric field enhancement, thereby improving the detection sensitivity and imaging contrast in the early stage of lesion formation and when the lesion is small.

[0036] Example 1: Synthesis of SP11, an organic polymer molecule with acoustic afterglow 1. Specific Synthesis Steps: Tin compound D-1 and bromide A-1 were polymerized to prepare the organic polymer SP11 with acoustic afterglow. In the reaction system, the amount of both tin compound D-1 and bromide A-1 monomers was 0.21 mmol. Specifically, tin compound-1 (218 mg, 0.21 mmol) and bromide-1 (143 mg, 0.21 mmol) were added to a sealed, pressure-resistant three-necked flask reaction vessel, and 10 mL of anhydrous toluene was added to ensure complete dissolution or uniform dispersion of the reactants. The reaction system was then evacuated and purged with nitrogen three times to remove oxygen and establish a nitrogen-protected environment. A reflux apparatus was installed, and under nitrogen protection, tetrakis(triphenylphosphine)palladium (9 mg, 0.01 mmol) was added to the reaction system. The reaction system was heated in an oil bath to 120 °C and continuously stirred for 24 hours. After the reaction was complete, the reaction system was allowed to cool naturally to room temperature. 200 mL of methanol was slowly added to the resulting reaction solution to allow the polymerization product to precipitate completely. The solid was collected by filtration and washed with methanol to remove some residual solvent and small molecule impurities. Subsequently, the solid was placed in a Soxhlet extractor and subjected to Soxhlet extraction sequentially with methanol, n-hexane, acetone, and chloroform, with each solvent extraction time being 12 hours. Methanol, n-hexane, and acetone extractions were used to remove unreacted monomers, catalyst residues, oligomers, and other small molecule impurities, while chloroform extraction was used to dissolve and collect the target polymer. The chloroform extract was collected, concentrated to an appropriate volume by rotary evaporation, and then slowly added dropwise to excess methanol to allow the target polymer to precipitate again. The precipitate was collected by filtration, washed thoroughly with methanol, and then dried under vacuum to constant weight to obtain the afterglow organic polymer SP11.

[0037] 2. ¹H NMR spectroscopy: The acoustic afterglow organic polymer SP11 prepared in Example 1 was used as a solvent to prepare a 5 mg / mL test solution, with each solution having a volume of 550 μL. The test solution was transferred to an NMR tube, and ¹H NMR spectroscopy was performed using a nuclear magnetic resonance spectrometer. Figure 3 As shown, SP11 exhibits characteristic proton signals corresponding to the expected molecular structure. Since the obtained compound is a conjugated polymer, some proton signals show a certain degree of peak broadening, which is consistent with the typical characteristics of polymer nuclear magnetic resonance hydrogen spectrum, indicating that the polymerization reaction proceeded smoothly and confirming that the acoustic afterglow organic polymer SP11 has been successfully prepared.

[0038] Example 2: Synthesis and Characterization of SPBT-MMP-IRQ, a Nanoprobe Based on MMP-2 Activated Electric Field Enhanced Acoustic Peripheral. 1. First, maleimide-modified electro-acoustic afterglow nanoprobes SPBT-Mal-NPs based on the acoustic afterglow organic polymer SP11 and the piezoelectric component BTO were prepared. SP11 (100 μg), BTO (100 μg), and DSPE-PEG2000-Mal (2.5 mg) prepared in Example 1 were dissolved or dispersed together in 1 mL of tetrahydrofuran. Then, a mixture of 9 mL of water and tetrahydrofuran at a volume ratio of 9:1 was added, and the mixture was ultrasonically dispersed for 30 min under ice bath conditions to allow the components to self-assemble into nanoparticles. The tetrahydrofuran was removed by rotary evaporation to obtain SPBT-Mal-NPs. Using the same method but without adding BTO, control probes SP-Mal-NPs without the piezoelectric component were prepared; subsequently, an MMP-2 responsive peptide was coupled. 6.92 mg of the thiol-terminated MMP-2 cleavable peptide NH2-GPLGIAGC-SH and 0.89 mg of tris(2-carboxyethyl)phosphine were weighed and dissolved together in 10 mL of a 1:4 mixture of dimethyl sulfoxide and water. The mixture was stirred at 37 °C for 60 min. SPBT-Mal-NPs were prepared into a 0.1 mg / mL dispersion. 1 mL of this dispersion was added to the above peptide solution, and the mixture was stirred at room temperature in the dark for 5 h. The MMP-2 responsive peptide was coupled to the surface of the nanoprobe via a thiol-maleimide Michael addition reaction. After the reaction, the mixture was centrifuged and ultrafiltered, and washed successively with a 1:4 mixture of dimethyl sulfoxide and water, and then with deionized water or phosphate buffer to obtain SPBT-MMP-NH2 with a free amino terminus. The control probe SP-MMP-NH2 was prepared using SP-Mal-NPs according to the same method. SPBT-MMP-NH2 or SP-MMP-NH2 was prepared into a dispersion based on an SP11 concentration of 30 μg / mL, and 1 mL was used for later use. An NHS-IRQ solution was prepared using a 1:4 volume ratio of dimethyl sulfoxide and water to achieve a final NHS-IRQ concentration of 0.15 mmol / L in the reaction system. The above nanoprobe dispersion was added to the NHS-IRQ solution, and the reaction was carried out at room temperature in the dark with stirring for 5 hours. The IRQ quenching group was coupled to the end of the MMP-2 responsive peptide via an amidation reaction between the NHS ester and the free amino group. After the reaction, ultrafiltration purification was performed, and the nanoprobe was repeatedly washed with a 1:4 volume ratio of dimethyl sulfoxide and water, followed by deionized water or phosphate buffer, to obtain the MMP-2 activated electric field-enhanced acoustic afterglow nanoprobe SPBT-MMP-IRQ and the control probe SP-MMP-IRQ without piezoelectric components, respectively. The obtained nanoprobes were stored at 4 °C in the dark.

[0039] 2. UV-Vis-NIR Absorption and Fluorescence Emission Spectroscopy Tests: The nanoprobe prepared in Example 2 was diluted with deionized water to a concentration of SP11 of 30 μg / mL, with each sample volume being 2 mL. Deionized water was used as a blank control. The absorption spectrum was measured in the 300-1100 nm range using a UV-Vis-NIR spectrophotometer, and the fluorescence emission spectrum was measured in the 300-1500 nm range using a fluorescence spectrometer. Figure 4 As shown, SPBT-MMP-NH2 without the quenching group exhibits the characteristic absorption peak of SP11 at approximately 608 nm and produces significant fluorescence emission in the 650-900 nm range; NHS-IRQ has a characteristic absorption peak at approximately 764 nm. SPBT-MMP-IRQ retains both the characteristic absorption peak of SP11 at approximately 608 nm and the characteristic absorption peak of IRQ at approximately 764 nm, and its fluorescence emission signal is significantly lower than that of SPBT-MMP-NH2, indicating the successful preparation of SPBT-MMP-IRQ.

[0040] 3. Transmission electron microscopy and particle size measurement: 2 mL of the SPBT-MMP-IRQ prepared in Example 2 was diluted with ultrapure water to form a solution with an SP11 concentration of 30 μg / mL. 5 μL of the nanoprobe suspension was dropped onto the surface of a copper mesh and allowed to air dry before imaging with a transmission electron microscope. 1 mL of the nanoprobe was placed in a cuvette, and its particle size was measured using a nanoparticle size potentiometer. Figure 5 As shown, SPBT-MMP-IRQ is a uniformly distributed sphere; dynamic light scattering measurements indicate that the hydrodynamic particle size of SPBT-MMP-IRQ is approximately 79.9 nm.

[0041] 4. Piezoelectric Performance Testing: SPBT-MMP-IRQ prepared in Example 2 was dissolved in deionized water to form a dispersion with a concentration of 1 mg / mL. 20 μL of the sample was dropped onto a clean gold-plated silicon wafer, spread evenly, and dried under vacuum for 12 hours to obtain a nanoprobe film. A voltage acquisition device was then constructed by covering the film with conductive electrodes. The device was connected to a voltage acquisition apparatus, and the voltage-time curve was recorded under open-circuit conditions. Without ultrasound application, the background voltage signal of the system was recorded; subsequently, ultrasound stimulation was applied to the device, and the voltage changes before, during, and after the ultrasound treatment were acquired in real time. Figure 6 As shown, no voltage was detected in the SPBT-MMP-IRQ without ultrasound. With ultrasound, the SPBT-MMP-IRQ generated a peak voltage of approximately 50–100 mV due to the presence of the piezoelectric component BTO.

[0042] Example 3: Fluorescence signal changes and afterglow signal changes after SPBT-MMP-IRQ MMP-2 response 1. Fluorescence response of SPBT-MMP-IRQ to different concentrations of MMP-2: 2 mL of SPBT-MMP-IRQ dispersion with a SP11 concentration of 30 μg / mL was incubated with MMP-2 at final concentrations of 0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 ng / mL in pH 7.4 buffer for 30 min, and then the fluorescence response was measured using a fluorescence spectrometer. Figure 7 As shown, with the increase of MMP-2 concentration, the fluorescence signal of SPBT-MMP-IRQ gradually recovered and enhanced, showing a clear concentration dependence. Based on the above response results, the subsequent acoustic afterglow experiment used SPBT-MMP-IRQ with an SP11 concentration of 30 μg / mL and MMP-2 with a concentration of 100 ng / mL as the test conditions.

[0043] 2. Fluorescence signal changes of SPBT-MMP-IRQ after treatment under different conditions: 2 mL of SPBT-MMP-IRQ dispersion with an SP11 concentration of 30 μg / mL was taken and treated as follows: at 2.5 W / cm 2 The ultrasonic power was applied for 60 seconds; then incubated with 100 ng / mL MMP-2 in pH 7.4 buffer for 30 minutes; after incubation with 100 ng / mL MMP-2 for 30 minutes, the mixture was further incubated with 2.5 W / cm². 2 The ultrasonic power was applied for 60 seconds. After processing, the fluorescence emission spectra of each group were measured using a fluorescence spectrometer. Data are expressed as mean ± standard deviation and were repeated three times. A two-tailed unpaired t-test was used to assess the significance of differences between the two experimental groups, with *** indicating p < 0.001. Figure 8 As shown, SPBT-MMP-IRQ produced almost no obvious fluorescence signal when only ultrasound was applied; after incubation with MMP-2, its fluorescence signal recovered significantly, indicating that quenching was relieved. Further ultrasound application resulted in a decrease in the instantaneous fluorescence signal compared to the MMP-2-only treatment group.

[0044] 3. Afterglow response of SPBT-MMP-IRQ to different concentrations of MMP-2: 2 mL of SPBT-MMP-IRQ dispersion with an SP11 concentration of 30 μg / mL was incubated with MMP-2 at final concentrations of 0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 ng / mL in pH 7.4 buffer for 30 min, followed by incubation at 2.5 W / cm². 2 The ultrasonic power was processed for 60 seconds, and the afterglow image was measured using an IVIS in vivo optical imaging system. Data are expressed as mean ± standard deviation, and were repeated three times. Figure 9As shown, with increasing MMP-2 concentration, the afterglow imaging signal of SPBT-MMP-IRQ gradually recovers and strengthens, exhibiting a clear concentration dependence.

[0045] 4. Changes in afterglow imaging of SPBT-MMP-IRQ after treatment under different conditions: 2 mL of SPBT-MMP-IRQ dispersion with an SP11 concentration of 30 μg / mL was taken and treated as follows: at 2.5 W / cm 2 The ultrasonic power was applied for 60 seconds; then incubated with 100 ng / mL MMP-2 in pH 7.4 buffer for 30 minutes; after incubation with 100 ng / mL MMP-2 for 30 minutes, the mixture was further incubated with 2.5 W / cm². 2 The ultrasonic power was processed for 60 seconds. After processing, the afterglow images of each group were measured using an IVIS in vivo optical imaging system. Data are expressed as mean ± standard deviation and were repeated three times. A two-tailed unpaired t-test was used to assess the significance of differences between the two experimental groups. *** indicates p < 0.001. Figure 10 As shown, when only ultrasound was applied and when only incubated with MMP-2, SP-MMP-IRQ and SPBT-MMP-IRQ produced almost no significant afterglow signal; after incubation with MMP-2 and further ultrasound, the afterglow signal was significantly recovered, and SP-MMP-IRQ produced only a weak afterglow signal, while SPBT-MMP-IRQ containing piezoelectric components produced a significantly enhanced afterglow signal.

[0046] 5. Peripheral imaging images of the selective responses of SPBT-MMP-IRQ to different biological enzymes and reducing substances: Take SPBT-MMP-IRQ dispersion (30 μg / mL, 1 mL), and add MMP-2, Caspase-3, Granulase B (GranB), Acetylcholinesterase (AChE), Alkaline phosphatase (ALP), Trypsin (Try), Reduced Nicotinamide Adenine Dinucleotide (NADH), Glutamate Dehydrogenase (GDH), or Nitroreductase (NTR) to a final concentration of 100 ng / mL, respectively. Incubate in pH 7.4 buffer for 60 minutes. Subsequently, at 2.5 W / cm 2 The ultrasound power was processed for 60 seconds, and after the ultrasound was stopped, the afterglow image was measured using an IVIS in vivo optical imaging system. Data are expressed as mean ± standard deviation, and the process was repeated three times. Figure 11 As shown, the MMP-2 treatment group produced a significantly enhanced acoustic afterglow signal, while the Caspase-3, GranB, AChE, ALP, Try, NADH, GDH and NTR treatment groups only showed a weaker background signal or no significant signal enhancement.

[0047] 6. Cytotoxicity results of the nanoprobes: Using 4T1 cells as a model, the cytotoxicity of SPBT-MMP-IRQ and the control group SP-MMP-IRQ was evaluated using the CCK-8 assay. The two nanoprobes were prepared into dispersions with SP11 concentrations of 0, 5, 10, 20, 30, and 50 μg / mL in RPMI 1640 medium. After sterilization by UV irradiation, the dispersions were added to 96-well plates, with five replicates per group and a blank control. Cells were cultured at 37°C and 5% CO2 for 24 h. The medium was discarded, and the cells were washed twice with PBS. Medium containing 10% CCK-8 reagent was added, and the cells were incubated in the dark for another 1-2 hours. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated. Data are expressed as mean ± standard deviation, and the assay was repeated five times. Figure 12 As shown, within the SP11 concentration range of 0–50 μg / mL, the survival rate of 4T1 cells in the SP-MMP-IRQ and SPBT-MMP-IRQ treatment groups was higher than 80%, and there was no significant difference compared with the control group. This indicates that the two nanoprobes have low cytotoxicity and good biocompatibility within this concentration range, and can be used for subsequent in vivo imaging studies.

[0048] Example 4: In vivo acoustic afterglow luminescence imaging of subcutaneous tumors in mice at different developmental stages based on SPBT-MMP-IRQ and control probes.

[0049] For acoustic afterglow luminescence imaging results of mice with subcutaneous 4T1 tumors at different developmental stages: 50 μL of cells with a cell count of 1×10⁶ were used. 6 4T1 cell suspensions were injected into the right hind leg of BALB / c mice to establish a subcutaneous breast cancer tumor model. On days 2, 4, 6, 8, and 10 post-inoculation, SPBT-MMP-IRQ or the control probe SP-MMP-IRQ (without piezoelectric components) was injected via the tail vein at a volume of 0.2 mL and a concentration of 2 mg / mL. After the probe reached the predetermined enrichment time of 24 h, the tumor area was subjected to ultrasound excitation under the same conditions: ultrasound power of 2.5 W / cm² and ultrasound duration of 60 seconds. Immediately after ultrasound was stopped, afterglow images of the tumor site were acquired using an IVIS spectral imaging system, and the images were analyzed using Living Image software. Data are expressed as mean ± standard deviation and were repeated three times. A two-tailed unpaired t-test was used to assess the significance of differences between the two experimental groups. Figure 13 It can be seen that on days 2-4 after tumor inoculation, both probe groups showed only weak background signals; as the tumor grew, the SPBT-MMP-IRQ group could detect obvious acoustic afterglow signals in the tumor area on day 6, while the SP-MMP-IRQ group could not detect lesions until day 10.

[0050] Example 5: In vivo acoustic afterglow luminescence imaging of different formation stages of micrometastatic lesions in mouse lungs based on SPBT-MMP-IRQ and control probes.

[0051] 1. Acoustic afterglow luminescence imaging results for different stages of 4T1 lung metastases: A 4T1 lung metastasis model was established using the tail vein inoculation method. Cells were injected in a volume of 50 μL with a cell count of 1×10⁻⁶. 6 4T1 cell suspensions were injected into BALB / c mice via tail vein, followed by an injection of 100 μL of PBS to establish a lung breast cancer metastasis model. At weeks 1, 2, 3, 4, and 5 of the metastasis process, SPBT-MMP-IRQ or the control probe SP-MMP-IRQ without piezoelectric components were injected via tail vein, with a volume of 0.2 mL and a concentration of 2 mg / mL. After the probe reached the predetermined enrichment time of 24 h, the lung region was subjected to ultrasound excitation under the same conditions: ultrasound power of 2.5 W / cm² and ultrasound duration of 60 seconds. Immediately after ultrasound was stopped, afterglow images of the mouse lung region were acquired using an IVIS spectral imaging system and analyzed. Data are expressed as mean ± standard deviation and were repeated three times. A two-tailed unpaired t-test was used to assess the significance of differences between the two experimental groups. Figure 14 It is evident that in normal lung tissue and during the first 1-2 weeks of early metastasis, both probe groups showed only weak background signals. As lung metastases formed and MMP-2 activity increased, clear, subtle lung afterglow signals could be detected in the SPBT-MMP-IRQ group as early as week 3, while weak signals did not appear in the SP-MMP-IRQ group until week 5.

[0052] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A MMP-2 activated electric field-enhanced acoustic afterglow nanoprobe, characterized in that, The nanoprobe comprises an organic polymer SP11 with acoustic afterglow luminescence properties; a piezoelectric component BTO capable of generating a piezoelectric response under ultrasonic mechanical stress; an amphiphilic polymer for coating and stabilizing the SP11 and BTO; a MMP-2 cleavable peptide and a quenching group IRQ; the SP11, BTO, and amphiphilic polymer are co-precipitated and self-assembled into composite nanoparticles, with the MMP-2 cleavable peptide and quenching group coupled to the surface; in the presence of MMP-2, the MMP-2 cleavable peptide is specifically cleaved, causing the quenching group to move away from the organic polymer luminescent component, thereby restoring the acoustic afterglow luminescence signal; under ultrasonic action, the piezoelectric component generates a local electric field and further amplifies the restored acoustic afterglow signal.

2. A method for preparing the MMP-2 activated electric field enhanced acoustic afterglow nanoprobe of claim 1, characterized in that, The process includes the following steps: dissolving or dispersing an organic polymer SP11 with acoustic afterglow luminescence properties, a piezoelectric component BTO, and an amphiphilic polymer with maleimide groups in an organic solvent; adding the resulting organic phase to an aqueous mixed medium for self-assembly; removing the organic solvent to obtain maleimide-modified electric field-enhanced acoustic afterglow nanoprobes SPBT-Mal-NPs; mixing MMP-2 cleavable peptides with free thiol and free amino groups with the SPBT-Mal-NPs, causing the free thiol groups to undergo an addition reaction with the maleimide groups; and purifying the mixture to obtain MMP-2-responsive electric field-enhanced acoustic afterglow nanoprobes SPBT-MMP-NH2; and mixing the SPBT-MMP-NH2 with an NHS-activated IRQ quencher, causing the IRQ quencher to undergo an amidation reaction with the free amino groups on the MMP-2 cleavable peptides; and purifying the mixture to obtain MMP-2-activated electric field-enhanced acoustic afterglow nanoprobes SPBT-MMP-IRQ.

3. The preparation method according to claim 2, characterized in that, The organic polymer SP11 with acoustic afterglow luminescence properties has the following structure: ； The preparation method is as follows: Under a nitrogen protective atmosphere, tin compound Sn-D-Sn, bromide Br-A-Br, and tetrakis(triphenylphosphine)palladium are added in a set ratio. The mixed solution is heated to 100~180 °C and stirred for 12-48 hours. After the reaction is completed, the mixture is filtered and purified, and dried to obtain the target product, namely compound SP11, with a degree of polymerization n of 2-1000.

4. The preparation method according to claim 2, characterized in that, The amphiphilic polymer with maleimide groups is DSPE-PEG2000-Mal, and the organic solvent is tetrahydrofuran.

5. The preparation method according to claim 2, characterized in that, The amounts of SP11, barium titanate, and DSPE-PEG2000-Mal are 100 μg, 200 μg, and 2.5 mg, respectively. The volume ratio of the organic phase to the aqueous phase in the mixed phase is 1:5 to 1:20, and the self-assembly treatment time is 10 to 30 min.

6. The preparation method according to claim 2, characterized in that, The MMP-2 cleavable peptide is NH2-GPLGIAGC-SH. In the presence of tris(2-carboxyethyl)phosphine, its terminal thiol group undergoes an addition reaction with the maleimide group on the surface of SPBT-Mal-NPs to form a thioether bond, resulting in SPBT-MMP-NH2 with a free amino group on the surface.

7. The preparation method according to claim 2, characterized in that, The SPBT-MMP-NH2 further undergoes an amidation reaction with an NHS-activated IRQ quencher to obtain SPBT-MMP-IRQ.

8. The preparation method according to claim 2, characterized in that, The amidation reaction was carried out in an aqueous organic mixed medium at a temperature of 20-40 °C for 1-8 hours. The concentration of NHS-IRQ in the reaction system was 0.05-0.30 mmol / L. After the reaction was completed, the mixture was purified by ultrafiltration.

9. An application of the nanoprobe according to claim 1, characterized in that, The nanoprobes are used to identify different stages of tumor lesions and micrometastases.

10. The application according to claim 9, characterized in that, The tumor lesion is selected from one or more of the following: subcutaneous breast cancer tumors and metastatic lung tumors.

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