Self-assembly aggregation-induced emission nano-probe for targeted therapy and real-time imaging of non-small cell lung cancer and application of self-assembly aggregation-induced emission nano-probe

Abstract

The invention discloses a self-assembly aggregation-induced emission nanoprobe for targeted therapy and real-time imaging of non-small cell lung cancer and application, and belongs to the technical field of biological medicine. According to the invention, a self-assembled AIE compound with a structure of TPE-FFNPGYGKSLSRHDHIHHHPLGVR-PEG6 is firstly synthesized, the self-assembled AIE compound is self-assembled to form a self-assembled aggregation-induced emission nano-probe AIInps for targeting c-Met, and the AIInps has dual functions of realizing real-time imaging through fluorescence tracking, realizing targeted therapy by inhibiting the c-Met, realizing tumor specific accumulation and fluorescence tracking based on the design of the AIE, and realizing targeted treatment by inhibiting the c-Met. The AInps is a multifunctional NSCLC treatment platform, integrates targeted treatment and real-time imaging and has a very wide application prospect, and the limitation of poor tumor permeability and non-specific distribution in a traditional therapy method is overcome by the aid of the AInps, and the AInps is a multifunctional NSCLC treatment platform and integrates targeted treatment and real-time imaging.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a self-assembled aggregation-induced emission nanoprobes for targeted therapy and real-time imaging of non-small cell lung cancer and application. BACKGROUND

[0002] Lung cancer is the leading cause of cancer-related deaths in both men and women in the United States, with a five-year survival rate of approximately 20%. Despite the use of chemotherapy, surgical resection, and radiotherapy at various stages of NSCLC (non-small cell lung cancer), patient prognosis remains suboptimal, with a recurrence rate as high as 10%. Over 85% of lung cancer cases belong to the NSCLC type. The poor efficacy is mainly attributed to the fact that traditional chemotherapy drugs are more easily absorbed by normal tissues, making it difficult to penetrate the NSCLC tumor area. Studies have shown that only about 5% to 10% can reach the tumor tissue. At the same time, NSCLC has a high degree of invasiveness, showing strong invasiveness to organs and metastasis to the brain, lymph nodes, liver, bone, meninges, and bone marrow. Despite the progress in treatment strategies for NSCLC, early detection and precise localization of tumors remain key challenges in improving patient outcomes. One of the main obstacles to effective treatment is the accurate and real-time identification of tumor boundaries, which is crucial for diagnosis and surgical intervention. Current diagnostic methods mainly rely on imaging techniques such as CT, MRI, and PET scans, as well as pathological analysis. However, these methods often lack the precision required for intraoperative tumor localization, especially for small, deep, or poorly defined tumors. The resolution limitations of imaging techniques can hinder the accurate visualization of tumors, increasing the risk of incomplete resection or accidental removal of healthy tissue during surgery. Traditional tumor marking methods, such as metal particles or dyes, are not always effective for small, difficult-to-access, or highly mobile tumors. Therefore, there is an urgent need for new technologies that can achieve precise, real-time tumor localization during surgery to minimize the likelihood of residual tumor tissue and improve long-term survival rates for patients. Recent advances in molecular imaging and targeted therapy have opened up new avenues for improving tumor detection. In particular, the development of tumor-specific biomarkers and the application of targeted imaging agents that bind to these markers have the potential to improve the accuracy of diagnostic imaging and enable real-time monitoring of tumor dynamics during surgery. At the same time, fluorescence-guided surgery and other optical techniques show potential in improving the accuracy of intraoperative tumor localization.

[0003] C-Met is a receptor tyrosine kinase encoded by the MET proto-oncogene, which is the cell surface receptor for hepatocyte growth factor (HGF). The c-Met / HGF axis is aberrantly activated in a variety of cancers through mechanisms such as MET genomic amplification, transcriptional upregulation, and ligand dependence, leading to tumor progression, angiogenesis, increased invasiveness, enhanced metastatic potential, and increased drug resistance. In NSCLC, approximately two-thirds of patients have mutations in oncogenic drivers, including key signaling pathways such as PI3K / AKT / mTOR and Ras-Raf-MEK-ERK (MAPK), which are often over-activated. Mutations in genes such as EGFR, KRAS, BRAF, ALK, and ROS1 can lead to abnormal activation of these pathways, thereby driving malignant progression. Targeted therapies against these mutations have shown good efficacy in the early stage, but drug resistance remains a major obstacle to sustained clinical benefit. Drug resistance can be due to intrinsic mechanisms present at the time of diagnosis, as well as acquired mechanisms that develop during treatment. One of the main drivers of acquired drug resistance in NSCLC is the activation of alternative signaling pathways, particularly through the c-Met pathway. Abnormal c-Met signaling can result from somatic mutations, MET gene amplification, or overexpression of the c-Met receptor, and this pathway not only drives tumor initiation and progression, but also plays a key role in bypassing the effects of targeted therapies against EGFR or ALK. For example, in cases of resistance to EGFR inhibitors such as gefitinib and erlotinib, amplification of c-Met often appears as a compensatory mechanism, allowing the tumor to continue to grow and metastasize. Similarly, in the presence of ALK inhibitors, MET-driven signaling can activate survival pathways. In addition, although several drugs targeting c-Met such as crizotinib, cabozantinib, and tepotinib have been approved for the treatment of advanced or metastatic NSCLC, there are still issues with efficacy, selectivity, and safety. The high frequency of genetic mutations and the emergence of resistance mechanisms have led to widespread interest in combination therapies that simultaneously target multiple pathways. The use of c-Met inhibitors in combination with EGFR or ALK inhibitors has shown potential to overcome drug resistance, but issues such as toxicity, treatment cycles, and tumor heterogeneity remain. Therefore, the development of new generations of inhibitors and drugs that can more specifically target c-Met with fewer side effects is key to advancing personalized medicine for NSCLC.

[0004] Aggregation-induced emission luminogens (AIEgens) exhibit high brightness, excellent photostability, enhanced fluorescence intensity and the ability to generate reactive oxygen species (ROS) in the aggregated state, making them ideal fluorescent materials for image-guided cancer therapy and diagnosis. Previous studies have shown that AIE-based formulations have great potential in photodynamic therapy (PDT) and / or photothermal therapy (PTT). However, its application still faces multiple challenges, including non-specificity, low stability and short circulation half-life, leading to insufficient tumor accumulation and potential side effects on normal tissues, and the surface properties still need to be optimized to endow AIE aggregates with higher specificity, longer circulation time and lower cytotoxicity, thereby promoting their penetration of biological barriers and enhancing their effectiveness in lung cancer therapy. SUMMARY

[0005] In view of this, the purpose of the present application is to provide a self-assembled aggregation-induced emission nano-probe for targeted therapy and real-time imaging of non-small cell lung cancer and its application. The self-assembled aggregation-induced emission nano-material AIEnps provided by the present application is used for targeting c-Met. AIEnps has a dual function: one is to achieve real-time imaging through fluorescence tracking, and the other is to achieve targeted therapy by inhibiting c-Met. The design based on AIE realizes tumor-specific accumulation and fluorescence tracking, overcoming the limitations of poor tumor penetration and non-specific distribution in traditional therapy. AIEnps is a promising multifunctional NSCLC treatment platform that integrates targeted therapy and real-time imaging.

[0006] In order to achieve the above purpose, the present application provides the following technical solutions: In a first aspect, the present application provides a self-assembled AIE compound, which has the structure of TPE-FFNPGYGKSLSRHDHIHHHPLGVR-PEG6, and TPE is tetraphenylethylene.

[0007] Based on the above technical scheme, further, the tetraphenylethylene and dipeptide phenylalanine (FF) provide strong hydrophobicity, NPGY is used as a lysosome targeting sequence, KSLSRHDHIHHH is a c-Met binding sequence, PLGVR is an MMP2 responsive enzyme cutting sequence, PEG6 provides a hydrophilic structure, the compound has an amphiphilic structure, can self-assemble to form a nanostructure, under the action of the high expression of MMP2 enzyme in the tumor microenvironment, after the hydrophilic PEG is removed by enzyme cutting, the hydrophobic TPE and FF interact with each other, self-assemble into nanofibers in situ, expose the c-Met binding site, combine with the tumor cell c-Met, induce the AIE effect to realize fluorescence tracing; subsequently, under the guidance of the NPGY sorting sequence, the peptide-c-Met protein complex is introduced into the lysosome for degradation, so as to realize the dual functions of tumor tracing and c-Met degradation, and enhance the anti-tumor effect.

[0008] In a second aspect, the present application provides a preparation method of the self-assembled AIE compound, comprising the following steps: (1) coupling Fmoc-NH-PEG6-COOH to the swollen polypeptide synthesis resin, and adding piperidine-containing DMF to remove the Fmoc protection; (2) taking NH2-PEG6 coupled on the polypeptide synthesis resin in step (1) as a starting point, sequentially connecting amino acid residues from the C-terminal to the N-terminal until the complete amino acid sequence is synthesized, and adding piperidine-containing DMF to remove the Fmoc protection; (3) adding TPE-NHS solution containing 1-10 equiv TPE-NHS according to the amount of peptide on the polypeptide synthesis resin, and reacting on a shaking table at room temperature for 1-5 h in the dark, then cutting and purifying to obtain the product.

[0009] Based on the above technical scheme, further, the resin in step (1) is Wang resin, and the Fmoc-NH-PEG6-COOH is first activated by HATU and DIPEA, and then coupled to the swollen polypeptide synthesis resin; the coupling time is controlled to be 100-150 min.

[0010] Based on the above technical scheme, further, the volume percentage of piperidine in the piperidine-containing DMF in step (1) is 15-25%.

[0011] Based on the above technical scheme, further, the specific process of step (2) is: after Fmoc-AA-OH is activated by OxymaPure and DIC, it is added to the polypeptide synthesis resin, and coupling is carried out at room temperature for 60-120 min; Fmoc protection is removed by adding DMF containing piperidine; the above cycle is repeated, and the amino acid residues are sequentially connected from the C-terminal to the N-terminal according to the sequence, until the complete amino acid sequence is synthesized; the volume percentage of piperidine in the DMF containing piperidine is 15-25%.

[0012] Based on the above technical scheme, further, the solvent of the TPE-NHS solution in step (3) is DMSO, the concentration is 1-10 mM, and 2-5 equiv of DIPEA is added.

[0013] Based on the above technical scheme, further, the composition of the cutting solution used for cutting in step (3) is: TFA, triisopropylsilane (TIS) and H2O, the volume ratio is 95:2.5:2.5, the addition amount is 5-20 mL / g resin, and the shaking is carried out at room temperature for 1-5 h.

[0014] In a third aspect, the present application provides a self-assembled aggregation-induced emission nanoprobes AIEnps, which are prepared from the self-assembled AIE compounds by a double emulsion method.

[0015] In a fourth aspect, the present application provides a preparation method of the self-assembled aggregation-induced emission nanoprobes AIEnps, which comprises the following steps: 1) 10-100 mg of PLGA and 0.1-0.5 mg of self-assembled AIE compounds are added to 1-5 mL of an organic solvent, and vortexed until completely dissolved to obtain an oil phase; 2-10 mg of MgCl2 is dissolved in 100-300 μL of ultrapure water to obtain an inner aqueous phase; 100-500 mg of PVA is dissolved in 5-20 mL of ultrapure water, and stirred until completely dissolved to obtain an outer aqueous phase; 2) 100-300 μL of the inner aqueous phase is slowly added to 1-3 mL of the oil phase, and ultrasonically treated to obtain a primary emulsion (W / O); 3) The primary emulsion (W / O) prepared in step 2) is quickly poured into the outer aqueous phase under stirring, and emulsified by ultrasonication to obtain a double emulsion (W / O / W), which is transferred to an open container, and the organic solvent is volatilized to remove to obtain solid nanoparticles.

[0016] Based on the above technical scheme, further, in step 1), the molar ratio of lactic acid and glycolic acid of the PLGA is 1:1; and the organic solvent is dichloromethane (DCM).

[0017] Based on the above technical scheme, further, the power of the ultrasonic in steps 2) and 3) is 50-200 W, and the ultrasonic time is 30-200 seconds.

[0018] Based on the above technical scheme, further, the stirring speed in step 3) is 500-1000 rpm.

[0019] In a fifth aspect, the present application provides the use of the self-assembled AIE compound and the self-assembled aggregation-induced emission nanoprobes AIEnps in the preparation of an anti-tumor drug or a real-time imaging reagent.

[0020] Based on the above technical scheme, further, the tumor includes lung cancer, gastric cancer, renal cancer and ovarian cancer.

[0021] Based on the above technical scheme, further, the lung cancer includes non-small cell lung cancer.

[0022] Based on the above technical scheme, further, the cell line of the non-small cell lung cancer is A549 cell.

[0023] Based on the above technical scheme, further, the drug further includes a pharmaceutically acceptable excipient.

[0024] Based on the above technical scheme, further, the pharmaceutically acceptable excipient includes a filler, a diluent, a binder, a disintegrant and an emulsifying agent.

[0025] Compared with the prior art, the present application has the following beneficial effects: The self-assembled nanoprobes AIEnps targeting c-Met of the present application combine the aggregation-induced emission (AIE) property with selective kinase inhibition, and are specially directed to the c-Met / HGF axis which causes 30-50% NSCLC metastasis and 70% acquired drug resistance cases. The experimental results show that the AIEnps have a dual function: (1) c-Met-mediated tumor inhibition through downstream pathways, and (2) real-time fluorescence tracking ability through microenvironment-responsive AIE activation, which effectively overcomes the problems of limited tumor penetration (<5% ID / g) and non-specific biodistribution of traditional nanotherapeutic drugs; the incorporation of AIE into the design of nanomaterials not only provides a platform for fluorescence tracing, but also enhances the tumor targeting ability of the material, which is expected to provide a new idea for precise diagnosis and treatment of tumors. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present application, the drawings involved in the embodiments will be briefly introduced below.

[0027] Figure 1 A schematic diagram of the self-assembled nanoprobes AIEnps for targeted treatment and real-time imaging of non-small cell lung cancer.

[0028] Figure 2 Characterization results of self-assembled nanoprobes AIEnps, in which, A is HPLC analysis of AIEnps purity; B is MALDI-TOF MS characterization of molecular weight; C is TEM image showing spherical morphology before enzymolysis (scale: 500 nm); D is TEM image showing nanofibers after enzymolysis (scale: 100 nm); E is hydrodynamic particle size distribution; F is surface zeta potential measurement; G is fluorescence intensity comparison in H2O and THF solvent systems.

[0029] Figure 3 In vitro anti-tumor efficacy evaluation results of self-assembled nanoprobes AIEnps, in which, A is CCK-8 method to evaluate the toxicity of AIEnps on A549 cells; B is IC 50 values of A549 cells calculated based on dose-effect curve; C is AIEnps cytotoxicity results on HEK293T cells; D is laser confocal imaging results of c-Met (green) and AIEnps (blue) co-localization; E is the effect of 60 μM AIEnps treatment (0 / 24 / 48 h) on c-Met expression; F is the effect of AIEnps treatment for 24 h (0-120 μM) on c-Met expression; *P<0.05, **P<0.01.

[0030] Figure 4 Verification results of self-assembled nanoprobes AIEnps regulating EMT process, in which, A is qPCR analysis of c-Met downstream pathway genes of A549 cells treated with 60 μM AIEnps for 24 h (n=3); B is the effect of AIEnps on E-cadherin and N-cadherin shown by EMT marker quantification (*p<0.05, **p<0.01); C is cytoskeleton structure shown by phalloidin-rhodamine staining (scale=10 μm); D is Transwell migration experiment (scale=100 μm); E is cell migration quantitative analysis (**p<0.01, n=3); F is colony formation experiment (scale=100 μm); G is colony formation quantitative analysis (**p<0.01, n=3. DETAILED DESCRIPTION

[0031] The application will be described in detail below with reference to the embodiments. However, the embodiments of the application are not limited thereto, and it is obvious that the embodiments described below are only part of the embodiments of the application, and other similar embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0032] Example 1 Synthesis of AIE compound A self-assembled AIE compound was designed and synthesized, and the structure thereof is TPE-FFNPGYGKSLSRHDHIHHHPLGVR-PEG6, in which tetraphenylethylene (TPE) and dipeptide phenylalanine (FF) provide strong hydrophobicity at one end, NPGY is used as a lysosome targeting sequence, KSLSRHDHIHHH is a c-Met binding sequence, PLGVR is an MMP2 responsive enzyme cutting sequence, and PEG6 provides a hydrophilic structure. The synthesis process of the self-assembled AIE compound comprises the following steps: S1. Resin pretreatment and first coupling of PEG6 1. Swell the resin: add the resin Wang resin to 5 times the volume of DCM, shake at room temperature for 60 min; dry and wash with anhydrous DMF 3 times.

[0033] 2. Perform amino acid coupling using Fmoc-NH-PEG6-COOH: prepare Fmoc-NH-PEG6-COOH (4.0 equiv) + HATU (3.5 equiv) + DIPEA (6 equiv) in a small amount of anhydrous DMF, activate for 1 min, add the resin, couple at room temperature for 120 min, and wash with DMF.

[0034] 3. Add 20% piperidine / DMF to remove Fmoc protection (2x5 min + 1x10 min), and after each step, dry and wash with DMF 5 times until the washing liquid is colorless.

[0035] S2. Solid-phase peptide synthesis starting from PEG6 (C→N extension to the N terminus of the peptide) 1. Fmoc deprotection: add 20% piperidine / DMF to remove Fmoc protection (2x5 min + 1x10 min), and after each step, dry and wash with DMF 5 times until the washing liquid is colorless.

[0036] 2. Each coupling round: activate Fmoc-AA-OH + OxymaPure + DIC in a minimum volume of DMF, add the resin, and couple at room temperature for 90 min.

[0037] 3. Repeat the above cycle, and sequentially connect the amino acids according to the sequence from the C terminus to the N terminus until the complete sequence is synthesized.

[0038] S3. Coupling of N-terminal TPE-NHS on the resin 1. Ensure that the N-terminal is a free alpha-amine (Fmoc-deprotected) and the side chain is still protected; wash with DMF and dry.

[0039] 2. Prepare TPE-NHS: freshly prepare 50 mM in anhydrous DMSO (protect from light).

[0040] 3. Dilute TPE-NHS solution into DMF (DMF:DMSO = 9:1), add 5 equiv TPE-NHS based on the amount of peptide on the resin, and add DIPEA 3 equiv (to ensure amine deprotonation).

[0041] 4. Shake at room temperature for 2 h in the dark.

[0042] 5. After reaction, wash 5 times with DMF, and perform cleavage / purification.

[0043] S4. Cleavage, precipitation, purification 1. Final wash: wash with DMF, DCM, and dry under suction, 30 min under vacuum.

[0044] 2. Prepare cleavage cocktail: TFA, triisopropylsilane (TIS), and H2O in a volume ratio of 95:2.5:2.5, add about 10 mL / g of resin, and shake at room temperature for 2 h.

[0045] 3. Filter, combine TFA stream, and wash the resin with a small amount of TFA twice; slowly drop the combined filtrate into 10x volume of cold MTBE to precipitate the polypeptide, and place at -20 °C for 60 min.

[0046] 4. Centrifuge at low temperature (4000 rpm, 10 min), discard the supernatant, and cold wash the precipitate 3 times; dissolve in a 50:50 volume ratio of water / CAN solvent (containing 0.1% TFA), and lyophilize to obtain the crude product.

[0047] 5. Purify by preparative RP-HPLC (C18), collect the target peak, and lyophilize, the target purity is ≥95%.

[0048] S5. Analysis and confirmation Liquid chromatography analysis confirms the purity of the AIE compound (A), while mass spectrometry analysis verifies the molecular weight of the AIE compound and confirms the synthesis efficiency (B). Figure 2 A), while mass spectrometry analysis verifies the molecular weight of the AIE compound and confirms the synthesis efficiency (B). Figure 2 B).

[0049] Example 2 Characterization of AIEnps AIEnps were prepared by double emulsion method and characterized by multiple analytical techniques.

[0050] S1. Solution preparation 1. Oil phase preparation: weigh 50 mg PLGA (50:50) and 0.5 mg AIE molecule (TPE derivative) into a glass vial, add 2 mL dichloromethane (DCM), vortex until the polymer and AIE molecule are completely dissolved, the solution should be clear.

[0051] 2. Inner aqueous phase preparation: weigh 6.0 mg MgCl2, dissolve in 200 μL ultrapure water.

[0052] 3. Outer aqueous phase preparation: weigh 200 mg PVA, dissolve in 10 mL ultrapure water, heat in a water bath at 80 °C and stir until the PVA is completely dissolved, the solution is clear. Cool to room temperature for use.

[0053] S2. Preparation of primary emulsion Slowly add 200 μL of the inner aqueous phase to 2 mL of the oil phase, immediately use a probe-type ultrasonic instrument (power 100 W) to ultrasonicate in an ice-water bath for 60 seconds to obtain a milky white primary emulsion (W / O).

[0054] S3. Preparation of double emulsion (W / O / W) 1. Pour the above prepared W / O into the outer aqueous phase (PVA solution) being stirred vigorously (500-1000 rpm) immediately.

[0055] 2. Continue to emulsify the entire mixture using a probe-type ultrasonic instrument in an ice-water bath (power 80 W) for 90 seconds, at this time a double emulsion (W / O / W) is formed, the system is milky white.

[0056] S4. Solvent evaporation and nanoparticle solidification Transfer the double emulsion to a larger open beaker and place it on a magnetic stirrer, stir at 300 rpm for 3 hours to allow the DCM to completely evaporate, as the solvent evaporates, the PLGA in the oil phase gradually solidifies, encapsulating the AIE molecule inside, forming solid nanoparticles (AIEnps).

[0057] S5. Collection and purification 1. Centrifugal washing: transfer the solidified nanoparticle suspension to a centrifuge tube, centrifuge at 15,000 rpm in a high-speed centrifuge for 30 minutes to precipitate the nanoparticles, carefully discard the supernatant and resuspend the precipitate with ultrapure water, centrifuge again, this washing step is repeated 3 times to completely remove PVA and unencapsulated substances.

[0058] 2. Final dispersion: resuspend the final precipitate with 2 mL of ultrapure water and filter through a 0.45 μm filter membrane to remove a small amount of aggregates that may be present.

[0059] 3. Storage: The well-dispersed AIEnps suspension can be stored at 4 °C in the dark. The addition of 5% trehalose as a lyoprotectant allows for freeze-drying to obtain a solid powder for long-term storage.

[0060] TEM analysis shows that AIEnps have a significant morphological change after MMP2 enzymolysis Figure 2 C, 100 nm scale; Figure 2 D, 500 nm scale). In FBS-containing DMEM medium (37 °C), the average particle size of AIEnps is 383.7 ± 46.57 nm Figure 2 E, and the surface zeta potential is -15.2 ± 3.86 mV Figure 2 F. To further study the transformation of AIE materials from water-soluble pre-enzymolysis conformation to lipid-soluble post-enzymolysis state, H2O and THF were used as solvents for fluorescence spectrum analysis. The comparison of fluorescence spectrum of AIE nanomaterials after enzymolysis in different solvent systems (RFP detection mode) is shown in Figure 2 G. Compared with the aqueous solution system, AIE nanomaterials in the post-enzymolysis conformation showed significantly enhanced fluorescence intensity, confirming the expected effect of enhanced emission intensity after enzymolysis.

[0061] Example 3 In vitro experimental evaluation of the anti-tumor efficacy of AIEnps To evaluate the tumor-killing performance of AIE nanomaterials, A549 lung adenocarcinoma cells were selected as the tumor model to evaluate the concentration-dependent cytotoxicity of AIEnps, and the specific experimental process is as follows: Lung adenocarcinoma cells A549 were seeded in a 96-well plate at a density of 1 × 10 4 / well, and after 6 h of culture, different concentrations of AIEnps (0-200 µM) were co-cultured for 24 h, 10 μl of CCK-8 buffer was added to each well, and incubated at 37 °C for 1 h. Then the absorbance at 450 nm was measured using a microplate reader (BIO RAD Company), and the cell survival rate was calculated.

[0062] Compared with the 0 µM control group, the A549 cell viability decreased in a concentration-dependent manner after 24 hours of AIEnps treatment, and a significant difference in viability occurred when the concentration was > 50 µM Figure 3 A). The IC 50 value of A549 cells was 61.25 µM, indicating a strong tumor-killing effect on A549 cells Figure 3 B). Using HEK293T cells as a control to evaluate tumor specificity, only when the drug treatment time was extended to 48 hours, 100 µM concentration produced significant toxicity to control cells Figure 3 C). This differential response pattern confirms tumor-selective targeting.

[0063] Next, A549 cells were treated with 60 µM AIEnps for 24 hours, and after fixation and permeabilization, immunofluorescence staining was performed, and images were collected using confocal microscopy after mounting. The results showed that A549 cells exhibited characteristic blue fluorescence, and the c-Met protein on the cell membrane was labeled using immunofluorescence. Parallel immunofluorescence staining showed membrane-bound c-Met receptors, and after treatment, fluorescence attenuation and cell morphological elongation were observed Figure 3 D), which suggested that AIEnps mediated c-Met inhibition through surface binding and was accompanied by morphological regulation. Next, A549 cells were co-incubated with AIEnps nanomaterials at a concentration of 60 µM for different time points (0, 24, 48 hours), and Western blotting (WB) was performed. Cell samples at different time points were collected, and the protein concentration was determined using the BCA method, followed by SDS-PAGE electrophoresis. After membrane transfer, blocking, and antibody incubation, chemiluminescence detection was performed, and the results showed that the c-Met protein content decreased over time, indicating that the inhibitory effect of nanomaterials on c-Met became more obvious over time Figure 3 E). Similarly, when A549 cells were co-incubated with different concentrations (0, 30, 60, 120 µM) of nanomaterials for the same time, a similar result was observed, i.e., dose-dependent c-Met reduction Figure 3 F).

[0064] Example 4 Mechanism study of AIEnps-mediated c-Met pathway regulation MAPK and STAT signaling pathways have been confirmed as classic downstream effectors of c-Met receptor tyrosine kinase in tumorigenesis. Therefore, we co-incubated A549 cells with 60 µM AIEnps (IC 50 equivalent dose) and quantified the dynamic expression of key pathway genes through time-course qPCR analysis (0 / 24 / 48 h). First, RNA was extracted from treated cells, and RNA quality was detected. After passing the quality control, cDNA was obtained through reverse transcription, and the PCR reaction system was prepared and detected.

[0065] The results are shown in Figure 4 A), time analysis showed that after 48 hours of treatment, FAK (92.8 ± 10.7%), MAPK (87.0 ± 13.1%), RAF (94.8 ± 0.8%), and STAT (92.0 ± 5.1%) signaling components were gradually inhibited (Figure 4A), which was consistent with the known regulation of c-Met.

[0066] To analyze the c-Met-mediated EMT plasticity, we monitored E-cadherin (CDH1, epithelial marker) and N-cadherin (CDH2, mesenchymal marker) by cadherin switching assay. A549 cells were treated with 60 µM AIEnps for 24 hours, and the results showed that E-cadherin expression increased by 2.08-fold (p = 0.021), while N-cadherin was inhibited by 67.0 ± 7.9% (p < 0.001), confirming mesenchymal-epithelial transition (MET) (Fig. 2A). Figure 4 B}.

[0067] In addition, since most EMT changes are accompanied by cytoskeletal changes, phalloidin-rhodamine staining was performed on the cytoskeleton for further analysis. A549 cells were co-cultured with 60 µM AIEnps for 24 hours, and the treated cells were fixed, 0.1-0.5% Triton X-100 (PBS prepared) was added, and the cells were permeabilized at room temperature for 5-10 minutes, 1% BSA (PBS prepared) blocking solution was added, and the cells were blocked at room temperature for 30 minutes, and then diluted phalloidin-rhodamine working solution was added for staining. The plates were placed in a humidified box and incubated at room temperature for 30-60 minutes in the dark. After incubation, the slides were washed. The prepared slides were observed under a laser confocal microscope or a fluorescence microscope. The results showed that under the working concentration, the cytoskeleton of the incubated cells was obviously fragmented and the density was significantly reduced, indicating that the drug strongly inhibited the synthesis of the cytoskeleton (Fig. 2B), which supported the inhibition of EMT. Figure 4 C}, EMT.

[0068] In addition, cell migration was evaluated by transwell experiment. The experimental group (AIEnps concentration for treating A549 cells was 60 µM) and the control group (same volume of normal saline treatment) were set up. First, A549 cell suspension with a density of 1 × 10 5 cells / mL was prepared, and the control group was treated with serum-free cell suspension in the upper chamber and medium containing 10% FBS in the lower chamber; the experimental group was treated with serum-free cell suspension containing AIEnps in the upper chamber and medium containing 10% FBS and the same concentration of AIEnps in the lower chamber; before cell inoculation, the experimental group was resuspended with serum-free medium containing AIEnps, and the control group was treated with the same volume of serum-free cell culture medium; and then the plates were placed in a 37°C, 5% CO2 incubator for 24 hours. After incubation, the cells were fixed with 4% PF and stained with 0.1% crystal violet staining solution, and finally counted and photographed. The transwell experiment showed that compared with untreated A549 cells, AIEnps treatment resulted in a 61.5 ± 10.7% (p < 0.001) reduction in the migration and diffusion ability of A549 cells, and the typical images showed a significant reduction in migration (Fig. 2D-E). Figure 4 D-E}.

[0069] To investigate the self-renewal capacity of tumor stem cells, a clonogenic assay was performed. First, a single cell suspension of A549 was prepared and counted. The seeding density was determined to be 500 cells / well (6-well plate) and the cells were incubated in complete medium with or without AIEnps or the same volume of saline at 37°C in a 5% CO2 incubator for 1 week until visible colonies were formed. After fixation and staining, the colonies were counted. The clonogenic assay showed that the self-renewal capacity of tumor stem cells was significantly impaired (inhibition rate 53.2 ± 2.7%, p < 0.001; IC 50 = 61.25 µM) Figure 4 F-G).

[0070] In summary, this study demonstrates that AIEnps, as potent c-Met inhibitors, can inhibit downstream oncogenic signals and reverse the metastasis-promoting EMT program.

[0071] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit it; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A self-assembled AIE compound, characterized in that, The structure is TPE-FFNPGYGKSLSRHDHIHHHPLGVR-PEG6, and TPE is tetraphenylethylene.

2. The method for preparing the self-assembled AIE compound of claim 1, characterized in that, The method comprises the following steps: (1) coupling Fmoc-NH-PEG6-COOH to the swelled polypeptide synthesis resin, and adding piperidine-containing DMF to remove the Fmoc protection; (2) taking the NH2-PEG6 coupled on the polypeptide synthesis resin in step (1) as a starting point, sequentially connecting amino acid residues from the C-terminal to the N-terminal until the complete amino acid sequence is synthesized, and adding piperidine-containing DMF to remove the Fmoc protection; (3) adding a TPE-NHS solution containing 1-10 equiv TPE-NHS according to the amount of the peptide on the polypeptide synthesis resin, reacting for 1-5 h, cutting, and then obtaining the product after purification.

3. The production method according to claim 2, characterized by, The resin in step (1) is Wang resin, the Fmoc-NH-PEG6-COOH is first subjected to HATU and DIPEA activation treatment, and then coupled to the swelled polypeptide synthesis resin; the coupling time is controlled to be 100-150 min; and the volume percentage of piperidine in the piperidine-containing DMF is 15-25%.

4. The production method according to claim 2, characterized by, The specific process of the connection in step (2) is as follows: after Fmoc-AA-OH is activated by OxymaPure and DIC, it is added to the polypeptide synthesis resin, coupled at room temperature for 60-120 min, piperidine-containing DMF is added to remove the Fmoc protection, and the above cycle is repeated, amino acid residues are sequentially connected from the C-terminal to the N-terminal according to the sequence until the complete amino acid sequence is synthesized; and the volume percentage of piperidine in the piperidine-containing DMF is 15-25%.

5. The preparation method according to claim 2, characterized in that, The solvent of the TPE-NHS solution in step (3) is DMSO, the concentration is 1-10 mM, and 2-5 equiv of DIPEA is added; the composition of the cutting solution used for cutting is TFA, triisopropylsilane (TIS) and H2O, the volume ratio is 95:2.5:2.5, the addition amount is 5-20 mL / g of resin, and the cutting condition is room temperature vibration for 1-5 h.

6. A self-assembled aggregation-induced emission nanoprobes (AIEnps) characterized in that, The self-assembled AIE compound of claim 1 is prepared by a double emulsion method.

7. The method for the preparation of self-assembled aggregation-induced emission nanoprobes AIEnps according to claim 6, characterized in that, The method comprises the following steps: 1) 10-100 mg of PLGA and 0.1-0.5 mg of the self-assembled AIE compound are added to 1-5 mL of an organic solvent, vortexed until completely dissolved to obtain an oil phase; 2-10 mg of MgCl2 is dissolved in 100-300 μL of ultrapure water to obtain an inner aqueous phase; 100-500 mg of PVA is dissolved in 5-20 mL of ultrapure water, stirred until completely dissolved to obtain an outer aqueous phase; 2) 100-300 μL of the inner aqueous phase is slowly added to 1-3 mL of the oil phase, ultrasonically treated to obtain a primary emulsion (W / O); 3) the primary emulsion (W / O) prepared in step 2) is quickly poured into the outer aqueous phase under stirring, ultrasonically emulsified to obtain a double emulsion (W / O / W), transferred to an open container, and the organic solvent is volatilized to remove to obtain solid nanoparticles.

8. The preparation method according to claim 7, characterized in that, The molar ratio of lactic acid and glycolic acid of the PLGA in step 1) is 1:1; the organic solvent is dichloromethane; the power of the ultrasonic in steps 2) and 3) is 50-200 W, and the ultrasonic time is 30-200 seconds; the stirring speed in step 3) is 500-1000 rpm.

9. Application of the self-assembled AIE compound in claim 1 or the self-assembled aggregation-induced emission nanoprobes AIEnps in claim 6 in the preparation of an antitumor drug or a real-time imaging reagent.

10. Use according to claim 9, characterized in that, The tumor includes lung cancer, gastric cancer, kidney cancer and ovarian cancer; the lung cancer includes non-small cell lung cancer.

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