DLL4-VEGF dual-targeting aggregation-induced emission nanoprobe, preparation method thereof and application of nanoprobe in tumor surgical navigation

Abstract

The invention relates to a DLL4-VEGF (vascular endothelial growth factor) dual-targeting aggregation-induced emission nanoprobe, a preparation method thereof and application of the DLL4-VEGF dual-targeting aggregation-induced emission nanoprobe in tumor surgical navigation, the DLL4-VEGF dual-targeting aggregation-induced emission nanoprobe comprises a VEGF target targeting peptide, a self-assembly polypeptide and a DLL4 target targeting peptide which are connected in sequence, and fluorescence imaging molecules are connected to the VEGF target targeting peptide. The probe can effectively permeate into a deep tumor area due to the small-size characteristic, so that high permeability is realized; meanwhile, DLL4 and VEGF overexpressed on the surfaces of cancer cells can be specifically combined, tumor boundaries and tiny metastases are accurately recognized, and the enrichment capacity of tumors at target sites is remarkably enhanced; meanwhile, the probe carries a fluorophore and is suitable for real-time fluorescence navigation of laparoscope / robot-assisted tumor resection; the probe exists in the form of nanoparticles in body fluid circulation, forms change and gather to form nanofibers when reaching a tumor microenvironment, and can realize long-acting retention of a target part, so that high-resolution fluorescence imaging and precise surgical navigation are realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and relates to a DLL4-VEGF dual-targeted aggregation-induced luminescence nanoprobe, a preparation method thereof, and an application thereof in tumor surgery navigation. Background Art

[0002] Renal cell carcinoma (RCC) is one of the common malignant tumors of the urinary system, and surgical resection (including partial nephrectomy and radical nephrectomy) is currently the main treatment for early-stage renal cancer. However, accurate identification of tumor boundaries and micrometastases during surgery remains a major challenge. Although traditional imaging examinations (such as CT and MRI) can provide preoperative tumor localization, they cannot display tumor tissue in real time and dynamically during surgery, resulting in positive margins or residual microlesions in some cases, affecting the surgical cure rate. In addition, excessive resection of normal renal tissue may impair renal function and affect the patient's quality of life after surgery. Therefore, the development of a navigation technology that can display tumor boundaries and microlesions in real time and with high resolution during surgery is crucial to improving the accuracy of renal cancer surgery.

[0003] In recent years, fluorescence imaging navigation technology has shown great potential in tumor surgery due to its advantages such as high sensitivity, real-time visualization and simple operation. Currently, the fluorescent probes commonly used in clinical practice (such as indocyanine green, ICG) mainly rely on the enhanced permeability retention effect (EPR effect) to enrich at the tumor site, but they have problems such as insufficient targeting, high background signal, and short retention time. It is difficult to accurately define the tumor edge, especially the detection rate of micrometastases is low. In addition, small molecule probes such as ICG are rapidly metabolized in the body and cannot meet the needs of long-term surgical navigation. Therefore, there is an urgent need to develop new targeted fluorescent probes to improve the accuracy and durability of intraoperative navigation.

[0004] Peptide nanomaterials have attracted considerable attention in tumor-targeted imaging and therapy due to their excellent biocompatibility, biodegradability, and ease of functional modification. However, most current peptide probes still face challenges such as insufficient targeting and short retention times, limiting their application in surgical navigation. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention aims to provide a DLL4-VEGF dual-targeted aggregation-induced luminescence nanoprobe, a preparation method thereof, and an application thereof in tumor surgical navigation.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention provides a DLL4-VEGF dual-targeted aggregation-induced luminescence nanoprobe, which includes a VEGF target targeting peptide, a self-assembling polypeptide and a DLL4 target targeting peptide connected in sequence, and a fluorescent imaging molecule is connected to the VEGF target targeting peptide.

[0008] Tumor angiogenesis is a key mechanism in the development and progression of many tumors. The VEGF (vascular endothelial growth factor) and DLL4 (Delta-like ligand 4) signaling pathways play a crucial role in the abnormal proliferation, invasion, and metastasis of tumor blood vessels. VEGF is a classic angiogenic factor, and its overexpression promotes increased tumor vascular permeability. The DLL4 / Notch signaling pathway regulates vascular branching and maturation. These two pathways work synergistically to form a highly disorganized tumor vascular network.

[0009] This invention utilizes a rationally designed DLL4-VEGF dual-targeted aggregation-induced luminescence nanoprobe. Its small size allows for efficient penetration deep into tumors, achieving high permeability. It also specifically binds to DLL4 and VEGF overexpressed on the surface of cancer cells, accurately identifying tumor boundaries and micrometastases, significantly enhancing tumor accumulation at the target site. Furthermore, the probe carries a fluorescent moiety, making it suitable for real-time fluorescence navigation during laparoscopic / robotic-assisted tumor resection. The probe circulates as nanoparticles in body fluids, undergoes a morphological transformation upon reaching the tumor microenvironment, and aggregates to form nanofibers, achieving long-term retention at the target site. This enables high-resolution fluorescence imaging and precise surgical navigation, providing new technological support for personalized treatment of renal cancer. Furthermore, the probe exhibits excellent biocompatibility and high safety.

[0010] Preferably, the self-assembling polypeptide is selected from any one or a combination of at least two of the following polypeptide sequences: GNNQQNY, KLVFF, FF, ITSVV, GFLG, DEVD, RVRR.

[0011] Compared with self-assembling polypeptides of other polypeptide sequences, the above-mentioned self-assembling polypeptides have a better ability to aggregate probes to form nanofibers, thereby achieving a better long-term retention effect at the target site.

[0012] Preferably, the VEGF target peptide is selected from any one or a combination of at least two of the following polypeptide sequences: RRKRRRK, HKHHKHHKHKHK, CTTHWGFTLC, and DLLPNEV.

[0013] Preferably, the DLL4 target peptide is selected from any one or a combination of at least two of the following polypeptide sequences: LFHLFIYIF, CRRHRQHHRC, VEPNCDIHVMW.

[0014] Preferably, the fluorescent imaging molecule is selected from any one or a combination of at least two of the following molecules or their derivatives: Cy5, Cy5.5, Cy7, FITC, BODIPY, and NBD.

[0015] In the present invention, the VEGF target peptide and the self-assembling polypeptide are bonded by an amide bond or connection, wherein n is selected from any integer of 3-6 (eg, n=3, n=4, n=5 or n=6).

[0016] In the present invention, the DLL4 target peptide and the self-assembling polypeptide are connected by an amide bond or connection, wherein n is selected from any integer of 3-6 (eg, n=3, n=4, n=5 or n=6).

[0017] In the present invention, the fluorescent imaging molecule is linked to the VEGF target peptide via an amide bond.

[0018] In a second aspect, the present invention provides a method for preparing the DLL4-VEGF dual-targeting aggregation-induced luminescence nanoprobe according to the first aspect, the preparation method comprising the following steps:

[0019] The free amino acids constituting the VEGF target targeting peptide, the self-assembling polypeptide and the DLL4 target targeting peptide are used as raw materials to synthesize polypeptide molecules by solid-phase synthesis, and then the fluorescent imaging molecule is linked to the VEGF target targeting peptide under alkaline conditions to obtain the DLL4-VEGF dual-targeting aggregation-induced luminescence nanoprobe.

[0020] The dual-targeted aggregation-induced luminescence nanoprobe of the present invention has a simple preparation process, is easy to operate, is suitable for industrial production, and is convenient for storage.

[0021] Preferably, the preparation method comprises the following steps:

[0022] (1) The first amino acid of the DLL4 target peptide was fixed on the resin, the N-terminus was Fmoc-protected, and then the resin was swollen;

[0023] (2) Using a deprotecting agent to remove the Fmoc-protected amino group, after repeated washing and detection, the next amino acid of the desired polypeptide sequence is added for full reaction, and the amino acids are connected in sequence to obtain a polypeptide sequence fixed on the resin;

[0024] (3) The obtained polypeptide sequence fixed on the resin is mixed with the fluorescent imaging molecule under alkaline conditions to shrink the resin, cleave the polypeptide sequence from the resin, and blow dry with nitrogen to obtain the dual-targeted aggregation-induced emission nanoprobe.

[0025] The peptide solid-phase synthesis method of the present invention uses C-terminal to N-terminal synthesis. The Kaiser test is used to detect coupling and deprotection groups (three solutions: A: 0.5 g of ninhydrin dissolved in 10 mL of anhydrous ethanol; B: 20 ​​g of phenol dissolved in 5 mL of anhydrous ethanol; C: 0.1 g of ascorbic acid dissolved in 5 mL of anhydrous ethanol). The test detects whether the Fmoc residue has been removed by the deprotecting agent. A dark purple resin in the test tube indicates successful removal of the protecting agent. When coupled to an amino acid, the resin becomes colorless in the presence of the Fmoc protecting group.

[0026] Preferably, the resin needs to be swelled with anhydrous DMF for 2-10 h, for example, 2 h, 2.5 h, 3 h, 4 h, 4.5 h, 5 h, 5.5 h, 6 h, 7 h, 7.5 h, 8 h, 8.5 h, 9 h, 9.5 h, 10 h, etc.

[0027] Preferably, the reaction in step (3) is carried out under light-proof conditions.

[0028] Preferably, the lysis is carried out in an ice-water bath for 2.5-3.5 h, for example, 2.5 h, 2.6 h, 2.7 h, 2.8 h, 2.9 h, 3 h, 3.1 h, 3.2 h, 3.3 h, 3.4 h, 3.5 h, etc.

[0029] Preferably, the nitrogen is blown dry and then washed with ether.

[0030] Other specific point values ​​within the above numerical range can be selected and will not be described in detail here.

[0031] In a third aspect, the present invention provides use of the DLL4-VEGF dual-targeted aggregation-induced luminescence nanoprobe according to the first aspect in preparing a probe for tumor diagnosis or treatment.

[0032] Preferably, the tumor comprises renal cell carcinoma.

[0033] In a fourth aspect, the present invention provides use of the DLL4-VEGF dual-targeted aggregation-induced luminescence nanoprobe according to the first aspect in preparing a probe for tumor surgical navigation.

[0034] Preferably, the tumor comprises renal cell carcinoma.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] This invention utilizes a rationally designed DLL4-VEGF dual-targeted aggregation-induced luminescence nanoprobe. Its small size allows for efficient penetration deep into tumors, achieving high permeability. It also specifically binds to DLL4 and VEGF overexpressed on the surface of cancer cells, accurately identifying tumor boundaries and micrometastases, significantly enhancing tumor accumulation at the target site. Furthermore, the probe carries a fluorescent moiety, making it suitable for real-time fluorescence navigation during laparoscopic / robotic-assisted tumor resection. The probe circulates as nanoparticles in body fluids, undergoes a morphological transformation upon reaching the tumor microenvironment, and aggregates to form nanofibers, achieving long-term retention at the target site. This enables high-resolution fluorescence imaging and precise surgical navigation, providing new technological support for personalized treatment of renal cancer. Furthermore, the probe exhibits excellent biocompatibility and high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the MALDI-TOF-MS verification image of the dual-targeted aggregation-induced emission nanoprobe in solution;

[0038] Figure 2 This is a schematic diagram of the dual-targeted aggregation-induced emission nanoprobe targeting tumor sites and self-assembly process in vivo;

[0039] Figure 3 This is the TEM verification image of the dual-targeted aggregation-induced emission nanoprobe triggered by different proteins;

[0040] Figure 4 This is a result verification diagram of the dual-targeted aggregation-induced luminescence nanoprobe specifically targeting tumor cells;

[0041] Figure 5 This is a diagram verifying the formation of β-sheet fibers by dual-targeted aggregation-induced emission nanoprobes at the cellular level;

[0042] Figure 6 This is a diagram verifying the experimental results of the long-term retention of dual-targeted aggregation-induced luminescence nanoprobes on target cells;

[0043] Figure 7 This is an in vivo imaging image of the distribution of dual-targeted aggregation-induced emission nanoprobes in mice;

[0044] Figure 8 This is the in vivo fluorescence quantitative statistical result of the distribution of dual-targeted aggregation-induced emission nanoprobes in mice;

[0045] Figure 9 This is the result of the in vitro human kidney perfusion experiment of the dual-targeted aggregation-induced luminescence nanoprobe. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0047] The Cy5 molecules involved in the following examples were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. with CAS No. 1032678-42-4.

[0048] Example

[0049] In this embodiment, a dual-targeted aggregation-induced emission nanoprobe (hereinafter referred to as SDVP-F) is constructed, whose chemical formula is RRKRRRK(Cy5)-(OEG)4-GNNQQNY-(OEG)4-LFHLFIYIF.

[0050] Its chemical structure is shown below:

[0051]

[0052] The preparation method is as follows:

[0053] (1) Swelling resin: Weigh 300 mg of Fmoc-Phe-Wang Resin resin into a solid-phase peptide synthesis tube, add N,N-dimethylformamide (DMF) activated with molecular sieves, and shake in a shaker to swell for 8 h;

[0054] (2) Deprotection: Wash once with 10 mL of DCM, then with DMF, alternating the washing steps (3 times), ending with the final wash with DMF. Add 10 mL of a deprotection agent (hexahydropyridine:DMF = 1:4) and place on a shaker for 15 min to remove the Fmoc group on the amino acid.

[0055] (3) Detection: Rinse repeatedly with DCM and DMF (3 times), then add one drop each of reagents A, B, and C and a small amount of resin to a centrifuge tube and heat in boiling water for 1 min. If the color of the resin turns purple, the Fmoc protecting group has been removed.

[0056] (4) Coupling: 10 times the equivalent of the amino acid loaded on the resin and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) were weighed and placed in 15 mL centrifuge tubes, and then 10 mL of coupling agent (DMF:N-methylmorpholine (NMM) = 19:1) was added and reacted for 10 minutes. The mixture was added to the peptide synthesis tube and reacted on a shaker for 2 hours.

[0057] (5) Detection: Repeatedly wash with DCM and DMF (3 times), then add Kaiser text detection reagent. If the color of the resin does not change, it proves that the amino acid is coupled;

[0058] (6) Cyclic coupling: Repeat steps (2), (3), (4), and (5) until all amino acids are completely coupled. After the last amino acid is completely coupled, use a deprotecting agent to remove the Fmoc protecting group;

[0059] (7) Coupling of Cy5 molecules: Weigh 5 equivalents of Cy5 and 10 equivalents of HBTU in a 15 mL centrifuge tube, then add 10 mL of coupling agent (DMF: N-methylmorpholine (NMM) = 19:1) and react for 10 min. Then add it to the peptide synthesis tube and react overnight.

[0060] (9) Shrinkage: Shrink the resin with methanol for 15 min, then transfer the shrunken resin to a 10 mL serum bottle and add a magnetic rod to the bottle;

[0061] (10) Lysis: Add 3 mL of lysis buffer (2.5% ultrapure water + 2.5% triisopropylsilane + 92.5% trifluoroacetic acid + 2.5% ethanedithiol), stir magnetically in an ice bath for 3 h, filter, rinse the serum bottle with TFA, blow dry with nitrogen, add it dropwise into ice ether, allow the polypeptide to precipitate in the ether, then transfer the polypeptide to a centrifuge tube, centrifuge at 4°C, 8000 r / min, repeat three times, then transfer the polypeptide to a 1.5 mL centrifuge tube, seal it with a sealing film, and allow the ether to evaporate overnight. Purify it using preparative reverse-phase HPLC to obtain a dual-targeted aggregation-induced emission nanoprobe.

[0062] The prepared dual-targeted aggregation-induced emission nanoprobe was characterized by mass spectrometry. The probe was dissolved in an aqueous solution (500 μg / mL) and a small amount of the solution was tested by mass spectrometry (MS, electrospray). Figure 1 As shown by Figure 1 It can be seen that the main peak of the mass spectrum is basically consistent with the molecular weight of the designed polypeptide material, which indicates that the target molecule has been synthesized, indicating that the dual-targeted aggregation-induced luminescence nanoprobe with the above structure has been successfully synthesized. The schematic diagram of the dual-targeted aggregation-induced luminescence nanoprobe targeting tumor sites in vivo and the self-assembly process is shown in the figure below. Figure 2 shown.

[0063] Comparative Example 1

[0064] In this comparative example, a dual-targeting nanoparticle (hereinafter referred to as DVP-F) was constructed. The chemical structure of DVP-F differs from that of SDVP-F only in that the self-assembling polypeptide GNQQNY is replaced by the polypeptide sequence GGGQGGY, while all other conditions remain unchanged. The preparation method thereof is described in the Examples.

[0065] Comparative Example 2

[0066] In this comparative example, a single-targeted aggregation-induced emission nanoprobe (hereinafter referred to as SDP-F) was constructed. The difference in chemical structure between the single-targeted aggregation-induced emission nanoprobe and SDVP-F is that the single-targeted aggregation-induced emission nanoprobe lacks the RRKRRRK modification, and its chemical formula is GNNQQNY-(OEG)4-KLFHLFIYIF.

[0067] Its chemical structure is shown below:

[0068]

[0069] The preparation method thereof is referred to the examples.

[0070] Comparative Example 3

[0071] In this comparative example, a single-targeted aggregation-induced emission nanoprobe (hereinafter referred to as SVP-F) was constructed. The only difference in its chemical structure from SDVP-F is the lack of LFHLFIYIF modification, and its chemical formula is RRKRRRK(Cy5)-(OEG)4-GNNQQNY.

[0072] Its chemical structure is shown below:

[0073]

[0074] The preparation method thereof is referred to the examples.

[0075] Test Example 1

[0076] TEM verification:

[0077] The dual-targeted aggregation-induced luminescence nanoprobe SDVP-F prepared in the example was dissolved in a pre-prepared PBS solution (200 μg / mL). 1 μL of VEGF protein (1 μg / μL) or 1 μL of DLL4 protein (1 μg / μL) was added, or both VEGF and DLL4 proteins were added simultaneously. The mixture was allowed to stand for complete assembly. A control group with no protein was used. 10 μL of each sample was dropped onto a copper mesh. After 10 minutes, the unadsorbed sample at the edge of the mesh was blotted with filter paper. The mesh was then stained with uranyl acetate solution for 2 minutes, washed twice with deionized water, blotted along the edge with filter paper, and allowed to stand until dry. Transmission electron microscopy was used for observation.

[0078] The results are as follows Figure 3As shown: the dual-targeted aggregation-induced luminescence nanoprobe of the present invention can form polypeptide nanofibers after contacting with VEGF protein and / or DLL4 protein.

[0079] Test Example 2

[0080] Verification of specific binding to tumor cells:

[0081] 786-O cells (renal cancer cells) and 293-T cells (normal kidney cells) were cultured at a rate of 1×10 4 Cells were seeded into a confocal microscopy dish overnight to allow attachment. SDVP-F, prepared in the example, was then added to the dish at a concentration of 30 μM and incubated for 20 minutes. The cells were then washed three times with PBS for 2 minutes each. The cells were fixed with 4% paraformaldehyde for 15 minutes and then washed three times with PBS. Hoechst dye was prepared according to the manufacturer's instructions and used for nuclear staining for 15 minutes. After three PBS washes, the dish was maintained in a solution of PBS and observed and photographed under a laser confocal microscope.

[0082] The results are as follows Figure 4 As shown in the figure, it can be seen that SDVP-F can specifically recognize 786-O renal cancer cells at 20 minutes and is almost invisible on negative cells 293-T, proving that the designed SDVP-F has the targeting ability to specifically recognize tumor cells.

[0083] Test Example 3

[0084] Verification of β-sheet formation at the cellular level:

[0085] Prepare two confocal microplates of 786-O cells, with 1 × 10 cells per dish. 4 cells. Place the confocal dish in an incubator and allow the cells to adhere overnight. Weigh the SDVP-F molecule prepared in the example and the DVP-F molecule prepared in the comparative example, configure them at a concentration of 30 μM, add them to the confocal dish and incubate for 20 minutes, absorb the solution and wash with PBS three times, each time for 2 minutes. Prepare NIAD-4 probe dye according to the instructions and stain the cells for 30 minutes (NIAD-4 is a fluorescent dye used to detect β-sheet structure in proteins). Wash with PBS three times, each time for 5 minutes. Add 4% paraformaldehyde to fix the cells for 15 minutes to maintain the cell morphology. After absorbing the paraformaldehyde and washing with PBS three times, add 1 mL of PBS solution to each confocal dish, place it under a laser confocal microscope for observation and photography.

[0086] The results are as follows Figure 5As shown in the figure, it can be seen that the dual-targeted aggregation-induced luminescence nanoprobe involved in the present invention specifically binds to the NIAD-4 probe on the surface of 786-O cells, showing yellow β-sheet fibers, proving that the SDVP-F probe can form β-sheet fibers on the surface of target cells, while the DVP-F molecule cannot form β-sheet fibers.

[0087] Test Example 4

[0088] Verification of long-term retention effect on target cells:

[0089] Prepare five confocal plates of 786-O cells, with 1 × 10 cells per dish. 4 cells. Place the confocal dish in an incubator and allow the cells to adhere overnight. Weigh the SDVP-F molecule prepared in the example and the DVP-F molecule prepared in the comparative example, configure them at a concentration of 30 μM, add them to the confocal dish and incubate for 60 minutes. After absorbing the solution, wash with PBS three times, each time for 2 minutes. After absorbing the PBS, add culture medium and continue culturing in the incubator. Take the time points of 0h, 6h, 12h, 24h, and 48h in sequence, place them under a laser confocal microscope for observation and photography. Nuclear staining is required before each photo: prepare Hoechst dye according to the instructions and stain the nucleus for 15 minutes.

[0090] The results are as follows Figure 6 As shown in the figure, it can be seen that the dual-targeted aggregation-induced luminescence nanoprobe of the present invention is still retained in the target area after 48 hours.

[0091] Test Example 5

[0092] Verification of long-term retention in mice:

[0093] Tumor cells 786-O were cultured until confluent, digested, centrifuged, and resuspended in 50 μL PBS per dish. 50 μL Matrigel was then added to obtain 100 μL cell suspension (5×10 6 100 μL of tumor cell suspension was subcutaneously inoculated into the hind limbs of mice (6-8 week old female BALB / c-nu athymic nude mice). When the tumor grew to 200 mm 3 The 786-O xenograft mouse model was successfully established.

[0094] Mice were randomly divided into 5 groups, and then 200 μL of SDVP-F, DVP-F, SDP-F, SVP-F, and ICG probe solutions were injected into the tail vein, with the fluorescent molecule concentration being 200 μM. The changes in fluorescence signals within 1, 2, 6, 12, 24, 48, and 72 h were monitored by a small animal imaging system. Figure 7 As shown; and the results were analyzed by fluorescence quantitative analysis, line graph Figure 8It is the fluorescence quantitative result of the tumor site.

[0095] As can be seen from the figure: at 72 hours, the SDVP-F probe is still retained in the tumor site. Compared with other control groups, it can efficiently target the tumor site and achieve long-term enrichment and retention effects.

[0096] Test Example 6

[0097] Targeting validation at human ex vivo kidney tumor sites:

[0098] The experimental protocol was approved by the Institutional Review Board of the Fourth Hospital of Harbin Medical University, and informed consent was obtained from the patients. In the operating room, isolated human kidney specimens were obtained immediately after nephrectomy. To ensure the viability of the renal tissue, the specimens were pretreated quickly after acquisition. The renal artery and renal vein were cannulated with vascular cannulas and connected to the flushing system. Subsequently, the kidneys were flushed with Ringer's lactate solution to remove residual blood and maintain tissue viability. The pretreated kidneys were connected to the circulating perfusion system to ensure that the system was well sealed and leak-free. Ringer's lactate solution containing oxygen and nutrients was continuously delivered to the kidneys through the perfusion system to simulate the physiological environment and maintain the metabolic activity of the renal tissue.

[0099] After confirming the proper function of the circulatory perfusion system, the tumor-bearing kidney was perfused with a Ringer's lactate solution containing 20 μM SDVP-F probe and ICG (indocyanine green) in a total volume of 350 mL at a rate of 30 mL / min for 2 hours. Following this, the kidney was rinsed with 4 L of fresh Ringer's lactate solution for 1 hour to remove unbound SDVP-F probe and ICG molecules. Following the rinse, the kidney specimen was stored at 4°C for subsequent fluorescence imaging. The tumor-targeting performance of SDVP-F was further verified by comparing the distribution of the SDVP-F probe with that of ICG.

[0100] The results are as follows Figure 9 As shown in the figure, it can be seen that compared with the ICG group, the SDVP-F probe has significant targeting to the tumor, with a clear boundary with normal tissue and a high signal-to-noise ratio.

[0101] The applicant declares that the present invention is illustrated by the above-described embodiments, but the present invention is not limited to the above-described embodiments. This does not mean that the present invention must rely on the above-described embodiments in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for raw materials in the present invention, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0102] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0103] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A DLL4-VEGF dual-targeting aggregation-induced luminescence nanoprobe, characterized in that: The DLL4-VEGF dual-targeted aggregation-induced luminescence nanoprobe comprises a VEGF target targeting peptide, a self-assembling polypeptide and a DLL4 target targeting peptide which are connected in sequence, and a fluorescent imaging molecule is connected to the VEGF target targeting peptide.

2. The DLL4-VEGF dual-targeting aggregation-induced luminescence nanoprobe according to claim 1, characterized in that: The self-assembling polypeptide is selected from any one or a combination of at least two of the following polypeptide sequences: GNNQQNY, KLVFF, FF, ITSVV, GFLG, DEVD, and RVRR.

3. The DLL4-VEGF dual-targeting aggregation-induced luminescence nanoprobe according to claim 1 or 2, characterized in that: The VEGF target peptide is selected from any one or a combination of at least two of the following polypeptide sequences: RRKRRRK, HKHHKHHKHKHK, CTTHWGFTLC, and DLLPNEV.

4. The DLL4-VEGF dual-targeting aggregation-induced luminescence nanoprobe according to any one of claims 1 to 3, characterized in that: The DLL4 target peptide is selected from any one or a combination of at least two of the following polypeptide sequences: LFHLFIYIF, CRRHRQHHRC, VEPNCDIHVMW.

5. The DLL4-VEGF dual-targeting aggregation-induced luminescence nanoprobe according to any one of claims 1 to 4, characterized in that: The fluorescent imaging molecule is selected from any one or a combination of at least two of the following molecules or their derivatives: Cy5, Cy5.5, Cy7, FITC, BODIPY, and NBD.

6. The DLL4-VEGF dual-targeting aggregation-induced luminescence nanoprobe according to any one of claims 1 to 5, characterized in that: The VEGF target peptide and the self-assembling polypeptide are connected by amide bonds or connection, wherein n is selected from any integer from 3 to 6; Preferably, the DLL4 target peptide and the self-assembling polypeptide are bonded by an amide bond or connection, wherein n is selected from any integer from 3 to 6.

7. The DLL4-VEGF dual-targeting aggregation-induced luminescence nanoprobe according to any one of claims 1 to 6, characterized in that: The fluorescent imaging molecule is connected to the VEGF target peptide through an amide bond.

8. The method for preparing the DLL4-VEGF dual-targeting aggregation-induced luminescence nanoprobe according to any one of claims 1 to 7, characterized in that: The preparation method comprises the following steps: The free amino acids constituting the VEGF target targeting peptide, the self-assembling polypeptide and the DLL4 target targeting peptide are used as raw materials to synthesize polypeptide molecules by solid-phase synthesis, and then the fluorescent imaging molecule is linked to the VEGF target targeting peptide under alkaline conditions to obtain the DLL4-VEGF dual-targeting aggregation-induced luminescence nanoprobe.

9. Use of the DLL4-VEGF dual-targeting aggregation-induced luminescence nanoprobe according to any one of claims 1 to 7 in the preparation of a probe for tumor diagnosis or treatment; Preferably, the tumor comprises renal cell carcinoma.

10. Use of the DLL4-VEGF dual-targeting aggregation-induced luminescence nanoprobe according to any one of claims 1 to 7 in the preparation of a probe for tumor surgical navigation; Preferably, the tumor comprises renal cell carcinoma.

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