Targeting probe for ESCC imaging and application thereof

Abstract

The invention relates to the technical field of biomedicine, in particular to a targeting probe for ESCC imaging and application of the targeting probe, the targeting probe is Cy5-double-targeting peptide, and the polypeptide sequence of the targeting probe is shown as SEQ ID NO: 1; the Cy5 <-> dual-targeting peptide has relatively high binding affinity to human ESCC cells in vitro. In vivo, optical imaging also verifies the tumor targeting ability of the Cy5-dual-targeting peptide in a subcutaneous ESCC tumor model carrying KYSE-30. Within several hours, the Cy5-double-targeting peptide shows a rapid tumor targeting capability and high-contrast imaging which lasts for 24 hours; the Cy5-dual-targeting peptide has the advantages of high selectivity and affinity, high sensitivity and specificity, assistance of early diagnosis and treatment decision, guidance of personalized treatment and noninvasiveness and noninvasiveness in ESCC imaging, and provides a more accurate, personalized and noninvasive method and means for clinical diagnosis and treatment of ESCC.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a targeting probe for ESCC imaging and application thereof. Background Art

[0002] Esophageal cancer is one of the most lethal malignancies, ranking sixth in cancer mortality worldwide. In Asian countries, esophageal squamous cell carcinoma (ESCC) is the main pathological type. Multimodal treatment based on extensive esophagectomy and lymph node dissection has significantly improved survival. Early ESCC has hidden symptoms, and most patients are not diagnosed until the late stage, which limits the option of radical treatment. Despite the low incidence of ESCC, its survival rate is extremely low, which highlights the primary importance of early treatment of ESCC. In response to the limited effectiveness of conventional white light endoscopy, new imaging systems have emerged.

[0003] Since biological tissues have very low absorption and autofluorescence within the near-infrared (NIR) spectrum (650-900 nm), near-infrared fluorescence (NIRF) imaging has been used to visualize various biomolecules closely related to disease processes at the cellular and even molecular levels.

[0004] A recent concept is to develop new molecular imaging methods using specific biochemical molecules that are altered during cancer progression. The emergence of some ESCC biomarkers may lead to new monitoring methods for identification and detection in high-risk populations. As an m6A-binding protein, human insulin-like growth factor 2 (IGF2) mRNA binding protein 2 (IGF2BP2) enhances mRNA stability in an m6A-dependent manner and is involved in the occurrence and development of various malignancies. As a tumor promoter, IGF2BP2 is significantly upregulated in EC tissues and is considered to be associated with poor prognosis. Given the high expression rate of IGF2BP2, molecular tracers targeting IGF2BP2 may help in the precise imaging of ESCC.

[0005] As a key factor in near-infrared fluorescence imaging, near-infrared imaging probes are usually composed of near-infrared dyes and targeting groups (including antibodies and their fragments, peptides, small molecules, etc.). Specific interactions occur with molecules during tumorigenesis and evolution. Peptides stand out among many targeting molecules because of their low immunogenicity, greater tissue penetration, faster blood clearance, and relatively simple production methods. Phage display technology combines the antigen recognition ability of recombinant proteins and is an effective screening system for the production of peptides or antibodies targeting specific molecules or tumor structures; therefore, it has great prospects in the field of monoclonal antibody and tumor-specific peptide development.

[0006] A targeted probe for ESCC imaging and its application are proposed, which is connected with a polypeptide of IGF2BP2 that can specifically bind to human ESCC and can have a significant targeting effect at the cellular level and in animal tumor models. Summary of the invention

[0007] The purpose of the present invention is to provide a targeting probe for ESCC imaging and its application. In vitro and in vivo experiments have confirmed that the Cy5-dual targeting peptide has high affinity, specificity and tumor targeting.

[0008] In order to achieve the above technical objectives and the above technical effects, the present invention is implemented through the following technical solutions:

[0009] A targeting probe for ESCC imaging, wherein the targeting probe is a Cy5-dual targeting peptide, and the polypeptide sequence thereof is shown in SEQ ID NO: 1.

[0010] Furthermore, in this embodiment, the method for preparing the above-mentioned targeting probe comprises the following steps:

[0011] S1: Solvent treatment: DMF and methanol were soaked in G3 molecular sieves overnight before use to remove impurities and water;

[0012] S2: Full swelling of the resin: weigh 2.0 g of blank Wang resin into a clean and dry reaction tube, add 15 mL of DMF, and activate at room temperature for about 30 min;

[0013] S3: Connect the first amino group: At room temperature, filter out the solvent from the previous step through a sand core, add 1mmol of the first amino acid at the C-terminus at a 5-fold molar excess, DMAP at a 5-fold molar excess, DIC at a 5-fold molar excess, and DMF as a solvent, and react at room temperature for 3 hours. After the reaction is completed, wash with DMF 4-6 times, 5-6mL each time. Then add an appropriate amount of pyridine and acetic anhydride in a volume ratio of 1:1, and react for 30 minutes. After the reaction is completed, wash with DMF 4-6 times, 5-6mL each time. (Function: to block the active micro-spots on the unreacted empty resin.)

[0014] S4: Removal of Fmoc protecting group: Remove the solvent from the previous step by suction, add 10 mL of 20% piperidine DMF solution to the resin, 2 After stirring for 10 min, the solution was filtered and 10 mL of 20% piperidine DMF solution was added. 2 Blow and stir for 5 minutes and then filter out the solution. Repeat this operation twice, then wash with DMF 4 times and methanol 2 times, 5-6 mL each time.

[0015] S5: Ninhydrin detection of removal effect: Take out a small amount of resin, wash it three times with methanol, add one drop of ninhydrin, KCN, and phenol solution, heat at 105-110℃ for 5 minutes, and it turns dark blue for a positive reaction, indicating that the removal is complete and the next step can be carried out; if it is colorless, it means that the protecting group is not completely removed, and the above deprotection operation needs to be repeated.

[0016] S6: Removal of the second amino acid and Fmoc protecting group: Weigh 3 times the molar excess of the second amino acid at the C-terminus, 3 times the molar excess of HBTU and 3 times the molar excess of HOBT in a reaction tube, add an appropriate amount of DMF solution to completely dissolve it, then add 10 times the molar excess of (pure) DIEA, react at room temperature for 40 minutes, and wash with DMF 4-6 times, 5-6 mL each time. Take a small amount of resin and detect it with ninhydrin detection reagent, which is colorless, then add 10mL20% piperidine DMF solution to remove Fmoc, and perform it twice, 10min and 5min respectively, then wash it with DMF 4 times and methanol 2 times, 5-6mL each time. Take out a small amount of resin and detect it with ninhydrin detection reagent. If it is blue, the next step of reaction can be carried out.

[0017] S7: Repeat step S6 in this way until the last amino acid at the N-terminus is synthesized, remove the Fmoc protecting group, and then drain.

[0018] S8: Resin removal and pure product separation and detection: Finally, use trifluoroacetic acid cutting solution (95% TFA: 2% TIS: 2% EDT: 1% H 2 O) cut for 2h, filter the reaction solution to obtain a trifluoroacetic acid solution of the polypeptide, blow the lysate dry with nitrogen as much as possible, precipitate with ether, centrifuge, and then wash with ether 3-5 times to obtain a white solid, dissolve it in pure water, desalt and purify it by HPLC, freeze-dry and precipitate crystals to obtain Cy5-dual targeting peptide, and take a small amount for MS analysis.

[0019] In another aspect, the present invention provides the application of the above-mentioned targeted probe in ESCC imaging.

[0020] Beneficial effects of the present invention:

[0021] The present invention identifies a peptide target of IGF2BP2, which is confirmed to be highly expressed in ESCC cell lines or tumor tissues and can be used as an imaging target for ESCC. The proposed Cy5-dual targeting peptide has a high binding affinity to human ESCC cells in vitro. In vivo, optical imaging also verified the tumor targeting ability of the Cy5-dual targeting peptide in a subcutaneous ESCC tumor model carrying KYSE-30. Within a few hours, the Cy5-dual targeting peptide showed rapid tumor targeting ability and high-contrast imaging that lasted for 24 hours. Specifically, it has the following features:

[0022] Beneficial effects:

[0023] 1. High selectivity and affinity: The design of Cy5-dual targeting peptide is based on a deep understanding of ESCC-related targets. One of the targeting peptide sequences has the ability to bind to the IGF2BP2 protein with affinity, and the other targeting peptide sequence can specifically bind to other proteins related to tumor growth and metastasis. The bidirectional targeting peptide can bind to multiple targets at the same time, enhancing the targeting of ESCC tissues. This dual targeting strategy improves the binding affinity and specificity of the probe to tumor cells. The amino acid residues in the targeting peptide sequence interact with the specific domains of the target protein to form a stable ligand-receptor complex. This high selectivity and affinity ensures the specific binding of the Cy5-dual targeting peptide to ESCC-related targets, improving the accuracy and reliability of imaging.

[0024] 2. High sensitivity and specificity: The multidirectional targeting strategy of Cy5-dual targeting peptide enables it to bind to multiple ESCC-related targets at the same time and enhance the interaction with tumor tissue. This multi-targeting strategy can increase the generation of imaging signals and the binding strength with tumor tissue, thereby improving the sensitivity of imaging. In addition, the design of Cy5-dual targeting peptide takes into account the possible differences in target expression between different ESCC cases. By combining multiple targets, Cy5-dual targeting peptide can reduce the occurrence of false positive results and increase the specificity of imaging. In addition, the near-infrared fluorescent dyes (such as Cy5) commonly used in Cy5-dual targeting peptides have a high optical penetration depth and low light scattering in the near-infrared spectral range, which can better penetrate biological tissues and improve the sensitivity of imaging. This multi-directional targeting strategy can not only improve the sensitivity of imaging, but also accurately identify and locate tumor tissues.

[0025] 3. Assistance in early diagnosis and treatment decision-making: Cy5-dual targeting peptide imaging can help doctors detect ESCC lesions early, assist in early diagnosis and treatment decision-making. The luminescent signal specifically bound by the targeted peptide can mark and visualize ESCC tumor tissue, allowing doctors to clearly observe the location, size and distribution of lesions. This information can help doctors determine the appropriate treatment plan, such as the scope of surgery and the need for lymph node dissection, and decide the timing of treatment. In addition, Cy5-dual targeting peptide imaging can also provide important basis for the staging and grading of lesions, which helps to formulate a more accurate treatment plan.

[0026] 4. Guidance for personalized treatment: Cy5-dual targeting peptide imaging provides information on the molecular characteristics and biological behavior of ESCC tumors, providing a basis for the selection of personalized treatment strategies. Through imaging, doctors can evaluate the expression levels of targets in ESCC cases and understand the molecular characteristics of tumors. This information can be combined with data from other clinical and molecular markers to provide a more comprehensive basis for the selection of personalized treatment plans. Describe and analyze the characteristics of tumor tissue from different angles to improve the accuracy and comprehensiveness of imaging. For example, combining MRI can provide more detailed tumor morphological information, and combining PET can provide more sensitive tumor metabolic information, making the imaging results more comprehensive and reliable.

[0027] 5. Non-invasive and non-invasive: Cy5-dual-targeted peptide imaging is a non-invasive and non-invasive imaging technology that does not require traumatic surgery or invasive procedures. During the imaging process, the patient only needs to receive an injection or take the targeted peptide and observe the luminescent signal of the tumor tissue through an external imaging device. This non-invasive imaging method does not cause additional pain or discomfort to the patient and allows for long-term monitoring and follow-up. Non-invasive imaging can provide dynamic information on pathophysiological changes and treatment effects, which helps to understand the development process of ESCC and guide the adjustment of treatment strategies.

[0028] In summary, Cy5-dual targeting peptide has the advantages of high selectivity and affinity, high sensitivity and specificity, assistance in early diagnosis and treatment decision-making, guidance of personalized treatment, and non-invasiveness and non-invasiveness in ESCC imaging. These effects are based on a deep understanding of ESCC and a comprehensive analysis of tumor characteristics, providing more accurate, personalized and non-invasive methods and means for the clinical diagnosis and treatment of ESCC.

[0029] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for describing the embodiments are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0031] Figure 1IGF 2BP 2 expression in ESCC cancer cell lines and tumor tissues. (A) Western blot of IGF 2BP2 in esophageal cancer cell lines. (B) Western blot of IGF 2BP 2 in esophageal cancer tumor tissues (T) and normal tissues (N). (C) IGFBP 2 mRNA expression in tumor tissues and normal tissues from 11 patients. **p<0.01. (D) Representative IHC staining images of IGF2BP 2 expression in ESCC tissues. -: negative staining, +: weak positive, ++: moderately positive, +: strongly positive. Magnification, 50 times, 200 times;

[0032] Figure 2 Identification of IGF2BP2 targeting peptides. (A) Flow chart for screening IGF2BP2 targeting peptides. (B) Amino acid sequence of IGF2BP2 protein. (C) Coomassie blue staining of IGF2BP2 protein after expression and purification. (D) Binding affinity of nine selected phages. OD values ​​were analyzed by phage ELISA;

[0033] Figure 3 This is a schematic diagram of the screening process described in an embodiment of the present invention;

[0034] Figure 4 This is a schematic diagram of the ELISA titer detection and positive retest according to the embodiment of the present invention;

[0035] Figure 5 The positive clone cross-reaction test described in the embodiment of the present invention;

[0036] Figure 6 This is a schematic diagram of MS analysis according to an embodiment of the present invention;

[0037] Figure 7 This is a schematic diagram of the HPLC analysis described in the embodiment of the present invention. DETAILED DESCRIPTION

[0038] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Example 1

[0040] The targeting probe for ESCC imaging described in this embodiment is a Cy5-dual targeting peptide, and its polypeptide sequence is shown in SEQ ID NO:1.

[0041] In this embodiment, the method for preparing the above-mentioned targeting probe comprises the following steps:

[0042] S1: Solvent treatment: DMF and methanol were soaked in G3 molecular sieves overnight before use to remove impurities and water.

[0043] S2: Full swelling of the resin: weigh 2.0 g of blank Wang resin into a clean and dry reaction tube, add 15 mL of DMF, and activate at room temperature for about 30 min;

[0044] S3: Connect the first amino group: At room temperature, filter out the solvent from the previous step through a sand core, add 1mmol of the first amino acid at the C-terminus at a 5-fold molar excess, DMAP at a 5-fold molar excess, DIC at a 5-fold molar excess, and DMF as a solvent, and react at room temperature for 3 hours. After the reaction is completed, wash with DMF 4-6 times, 5-6mL each time. Then add an appropriate amount of pyridine and acetic anhydride in a volume ratio of 1:1, and react for 30 minutes. After the reaction is completed, wash with DMF 4-6 times, 5-6mL each time. (Function: to block the active micro-spots on the unreacted empty resin.)

[0045] S4: Removal of Fmoc protecting group: Remove the solvent from the previous step by suction, add 10 mL of 20% piperidine DMF solution to the resin, 2 After stirring for 10 min, the solution was filtered and 10 mL of 20% piperidine DMF solution was added. 2 Blow and stir for 5 minutes and then filter out the solution. Repeat this operation twice, then wash with DMF 4 times and methanol 2 times, 5-6 mL each time.

[0046] S5: Ninhydrin detection of removal effect: Take out a small amount of resin, wash it three times with methanol, add one drop of ninhydrin, KCN, and phenol solution, heat at 105-110℃ for 5 minutes, and it turns dark blue for a positive reaction, indicating that the removal is complete and the next step can be carried out; if it is colorless, it means that the protecting group is not completely removed, and the above deprotection operation needs to be repeated.

[0047] S6: Removal of the second amino acid and Fmoc protecting group: Weigh 3 times the molar excess of the second amino acid at the C-terminus, 3 times the molar excess of HBTU and 3 times the molar excess of HOBT in a reaction tube, add an appropriate amount of DMF solution to completely dissolve it, then add 10 times the molar excess of (pure) DIEA, react at room temperature for 40 minutes, and wash with DMF 4-6 times, 5-6 mL each time. Take a small amount of resin and detect it with ninhydrin detection reagent, which is colorless, then add 10mL20% piperidine DMF solution to remove Fmoc, and perform it twice, 10min and 5min respectively, then wash it with DMF 4 times and methanol 2 times, 5-6mL each time. Take out a small amount of resin and detect it with ninhydrin detection reagent. If it is blue, the next step of reaction can be carried out.

[0048] S7: Repeat step S6 in this way until the last amino acid at the N-terminus is synthesized, remove the Fmoc protecting group, and then drain.

[0049] S8: Resin removal and pure product separation and detection: Finally, use trifluoroacetic acid cutting solution (95% TFA: 2% TIS: 2% EDT: 1% H 2 O) cut for 2h, filter the reaction solution to obtain a trifluoroacetic acid solution of the polypeptide, blow the lysate dry with nitrogen as much as possible, precipitate with ether, centrifuge, and then wash with ether 3-5 times to obtain a white solid, dissolve it in pure water, desalt and purify it by HPLC, freeze-dry to precipitate crystals, and take a small amount for MS analysis.

[0050] In another aspect, the present invention provides the application of the above-mentioned targeted probe in ESCC imaging.

[0051] Example 2

[0052] Expression of IGF2BP2 in cancer cell lines and tumor tissues

[0053] Immunoblotting was used to quantitatively analyze the expression of IGF2BP2 in ESCC cell lines and tumor tissues. Figure 1 As shown in A, esophageal cancer cell lines showed different degrees of IGF2BP2 overexpression, among which KYSE-30 had the highest expression among all esophageal cancer cells. Immunoblotting of patient-derived tumor tissues also showed that the expression of IGF2BP2 in tumor tissues was relatively higher than that in adjacent tissues ( Figure 1 B). The gene expression profiling analysis website of the GEPIA database (http: / / gepia.canner-pku.cn / , accessed on May 11, 2022) showed that the expression of IGF2BP2 was significantly increased in ESCC compared with normal tissues (Figure S1), which was also confirmed by the gene expression results of tumor and adjacent tissues of 11 patients with esophageal cancer ( Figure 1 C). To investigate the expression of IGF2BP2 in esophageal squamous cell carcinoma, immunohistochemistry was used to detect the expression of IGF2BP2 in 98 cases of esophageal squamous cell carcinoma tissues. The results showed that although the positive expression of IGF2BP2 in esophageal cancer tissues reached 96.9% (95 / 98) ( Figure 1 D), but the expression of IGF2BP2 was not correlated with clinical pathological parameters such as age, sex, tumor metastasis (TNM) stage and differentiation degree (Table 1). These results indicate that IGF2BP2 is an important tumor marker and is highly expressed in esophageal cancer tissues and cells, suggesting that IGF2BP2 may be a potential imaging target for esophageal cancer.

[0054] Identification of IGF2BP2-targeting peptides

[0055] Figure 2 A briefly describes the process of peptide screening. Based on the gene sequence of IGF2BP2, the amino acid sequence of IGF2BP2 protein was obtained ( Figure 2 B). IGF2BP2 protein was successfully purified, yielding a consistent predicted molecular weight of approximately 29 kDa, as shown by Coomassie blue staining ( Figure 2 C). A total of three rounds of cultivation and screening were performed. Significant enrichment of recovered phages was observed. After the last round of screening, 19 clones were randomly selected, verified by ELISA, and the phages were sequenced using high-throughput sequencing technology. The two highly repetitive polypeptide sequences showed a higher absorbance intensity at a wavelength of 450nm, which was significantly higher than that of the control group ( Figure 2 D). The high frequency of the peptide sequence indicates efficient enrichment during the screening process. The peptide sequence LSMPWSPTTYAS (called P12) had an OD value 10 times that of the control and was selected as the best candidate for further investigation, while a peptide with a scrambled sequence (called C12) was used as a control peptide.

[0056] Synthesis of IGF2BP2 targeting peptide

[0057] Synthesis steps

[0058] Solvent handling

[0059] DMF and methanol were soaked in G3 molecular sieves overnight before use to remove impurities and water.

[0060] Full swelling of the resin

[0061] Weigh 2.0 g of blank Wang resin into a clean and dry reaction tube, add 15 mL of DMF, and activate at room temperature for about 30 min.

[0062] First amino acid

[0063] At room temperature, filter out the solvent from the previous step through a sand core, add 1mmol of a 5-fold molar excess of the first amino acid at the C-terminus, a 5-fold molar excess of DMAP, a 5-fold molar excess of DIC, and DMF as a solvent, and react at room temperature for 3 hours. After the reaction is completed, wash with DMF 4-6 times, 5-6mL each time. Then add an appropriate amount of pyridine and acetic anhydride in a volume ratio of 1:1, and react for 30 minutes. After the reaction is completed, wash with DMF 4-6 times, 5-6mL each time. (Function: to block the active micro-spots on the unreacted empty resin.)

[0064] Removal of Fmoc protecting group

[0065] Remove the solvent from the previous step by suction, add 10 mL of 20% piperidine DMF solution to the resin, 2 After stirring for 10 min, the solution was filtered and 10 mL of 20% piperidine DMF solution was added. 2Blow and stir for 5 minutes and then filter out the solution. Repeat this operation twice, then wash with DMF 4 times and methanol 2 times, 5-6 mL each time.

[0066] Ninhydrin to detect removal effect

[0067] Take out a small amount of resin, wash it three times with methanol, add one drop each of ninhydrin, KCN, and phenol solution, heat at 105-110℃ for 5 minutes, and it turns dark blue for a positive reaction, indicating that the removal is complete and the next step can be carried out; if it is colorless, it means that the protecting group is not completely removed, and the above deprotection operation needs to be repeated.

[0068] Connect the second amino acid and remove the Fmoc protecting group

[0069] Weigh 3 times the molar excess of the second amino acid at the C-terminus, 3 times the molar excess of HBTU and 3 times the molar excess of HOBT in a reaction tube, add an appropriate amount of DMF solution to completely dissolve it, then add 10 times the molar excess of (pure) DIEA, react at room temperature for 40 minutes, and wash with DMF 4-6 times, 5-6 mL each time. Take a small amount of resin and detect it with ninhydrin detection reagent, which is colorless, then add 10 mL of 20% piperidine DMF solution to remove Fmoc, and perform it twice, 10 minutes and 5 minutes respectively, then wash it with DMF 4 times and methanol 2 times, 5-6 mL each time. Take out a small amount of resin and detect it with ninhydrin detection reagent. If it is detected as blue, the next step of reaction can be carried out.

[0070] By analogy, the steps of connecting the second amino acid and removing the Fmoc protecting group are repeated until the last amino acid at the N-terminus is synthesized, the Fmoc protecting group is removed, and then the mixture is drained.

[0071] Resin shedding and pure product separation detection

[0072] Finally, cut with trifluoroacetic acid cutting solution (95% TFA: 2% TIS: 2% EDT: 1% H2O) for 2 hours, filter the reaction solution to obtain trifluoroacetic acid solution of the polypeptide, blow the cleavage solution dry with nitrogen as much as possible, precipitate with ether, centrifuge, and then wash with ether 3-5 times to obtain a white solid. After dissolving in pure water, desalt and purify by HPLC, freeze-dry to precipitate crystals, and take a small amount for MS analysis.

[0073] Example 3

[0074] like Figure 6 Shown

[0075] MS analysis

[0076] Name: Cy5-dual targeting peptide

[0077] Peptide sequence: YHWYGYTPQNVI-GK(CY5)G-LSMPWSPTTYAS RRRRRRRR

[0078] Molar weight: 4994.79

[0079] Instrument: Shimadzu LCMS-2020

[0080] Probe: ESI

[0081] Nebulizer gas flow rate: 1.5L / min

[0082] Detector: 1.5kv

[0083] CDL: -20.0v

[0084] T.Flow: 0.2ml / min

[0085] CDL Temp.: 250℃

[0086] B.Conc.: 50%H2O / 50%ACN

[0087] Block Temp.:200℃

[0088] Certificate of Analysis

[0089] Name: Cy5-dual targeting peptide

[0090] Peptide sequencing: 5YHWYGYTPQNVI-GK(CY5)G-LSMPWSPTTYAS RRRRRRRR

[0091] Molar mass: 4994.79

[0092] Weight: 5mg

[0093] Storage: -4℃ or -20℃ (it is recommended to store at -20℃ or -80℃ after receiving the product)

[0094] Solubility: Normal

[0095]

[0096]

[0097] Example 4 Materials and Methods

[0098] Cells and mice

[0099] Human esophageal cancer cell lines EC-9706, EC-109, and TE-1 were obtained from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, while KYSE-30, KYSE-410, KYSE-450, and KYSE-520 were obtained from DSMZ (Germany, Germany). All cells were maintained in an incubator containing a humidified atmosphere of 5% carbon dioxide at 37°C. All cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin solution. Four-week-old mice were purchased from Guangdong Medical Experimental Animal Center (Guangdong, China). Subcutaneous tumor models were generated by subcutaneous injection of approximately 1 × 107 cells and 20% Matrigel suspension (Corning, New York, USA). All animal experiments were performed in accordance with protocols approved by the Animal Ethics Committee of the Fifth Affiliated Hospital of Sun Yat-sen University.

[0100] Peptide screening

[0101] like Figure 3-5 Shown

[0102] 1. Peptide library screening experiment (ELISA plate method)

[0103] 1. Coat the ELISA plate with 50ug / ml of the selected material in the middle, 100ul per well, and coat at 37℃ or 4℃ overnight.

[0104] 2. Discard the coating solution, wash with TBST 3 times, add 5% skim milk powder, block at 37℃ for 1h, and incubate 400ul per well.

[0105] 3. Discard the blocking solution, wash with TBST 3 times, add 50ul 5% skim milk powder and 50ul phage supernatant, and incubate at 37℃ for 2h

[0106] 4. Discard the supernatant, wash 6 times with 0.1% PBST, 1 min / time, and then wash 3 times with PBS, and spin dry the liquid each time.

[0107] 5. Elute with 100 μl 0.2 M Gly-HCl PH2.2 for 8 min, and neutralize with 15 μl 1 M Tris-HCl PH9.1;

[0108] 6. 10 μl of neutralization solution was diluted 1000, 10000, 100000 times and then infected with 1 ml of ER2738 (OD about 0.6) at 37°C for 5 min;

[0109] 7. After infection, add 3 ml of 50°C top culture medium, mix well, pour into 2YT / IPTG / Xgal medium and culture at 37°C overnight;

[0110] 8. Count the blue spots on the plate and calculate the number of recovered phages;

[0111] 9. Infect 40 ml of ER2738 (1:100 inoculation with shaken overnight) bacterial solution with the eluted phage at 37°C for 5 hours;

[0112] 10. Centrifuge at 12000g for 10 min;

[0113] 11. Take the supernatant and add 1 / 6 volume of 20% PEG / 2.5M NaCl at 4℃ for at least 2h;

[0114] 12. Centrifuge at 12000g for 15 min at 4°C and remove the solution;

[0115] 13. Add 1 ml of culture medium to resuspend the pellet;

[0116] 14. Repeat steps 1-13 for the second and third rounds of screening, adding 6 washes each time;

[0117] 2. Identification of positive clones

[0118] 1. After the third round of screening, the eluted phages were infected with E. coli, and then 3 ml of 50°C top culture medium was added, mixed and poured into 2YT / IPTG / Xgal medium and cultured at 37°C overnight;

[0119] 2. Pick up the blue spot monoclonal clone with a pipette tip and inoculate it into a 96-well plate;

[0120] 3. Incubate at 37°C for 5 hours and obtain phage by centrifugation;

[0121] 4. Coat the protein on the ELISA plate, 500 ng / well (depending on the situation);

[0122] 5. Block with 5% PBSM at 37°C for 1 h;

[0123] 6. Remove the blocking solution and add PBS to wash once;

[0124] 7. Add 50 μl of 5% PBSM and 50 μl of phage supernatant obtained in step 3 (or stock solution) to each well and incubate at 37°C for 1 h.

[0125] 8. Wash 3 times with TBST and 3 times with PBS;

[0126] 9. Add anti-P8 / HRP phage secondary antibody and incubate at 37°C for 40 min;

[0127] 10. Wash 3 times with TBST and 3 times with PBS;

[0128] 11. Add 100 μl TMB substrate and stop the reaction after 15 min in the dark.

[0129] 12. Read the results on the ELISA reader.

[0130] The positive clone detection is shown in the following table;

[0131]

[0132] During the phage screening process, the phage input amount is controlled, and the screening source is reduced in each round. In principle, the titer will increase after each round of panning, but in the actual panning process, it will vary due to different panning schemes, and the actual titer test results shall prevail. After 2-3 rounds of screening, single spots can be picked to detect whether there are positive clones that recognize the target bacteria but not the cross-bacteria.

[0133] A 12-peptide phage random peptide library was panned. During the panning, other irrelevant his-tagged colon proteins were first used for shielding, and then the target protein was used for 3-4 rounds of panning. The panning scheme was optimized during the panning stage, and it was performed twice, 3-4 rounds / time. After that, single clones were selected to induce phage for ELISA titer detection. Through retesting and cross-reaction testing, 19 positive single clones were finally selected for sequencing, and a total of 17 sequencing results and 9 sequences were obtained, among which sequences 1 and 2 were repeated, indicating that the enrichment of these two sequences was more obvious.

[0134] SDS-PAGE and western blotting

[0135] For cells and tumor tissues, proteins were extracted on ice using RIPA lysis buffer (P0013 E, Beyotime, Shanghai, China). Protein concentration was determined by BCA protein assay kit (P0012, Beyotitime, Shanghai, China) according to the manufacturer's instructions. After boiling at 98 ° C for 10 minutes, protein samples with loading buffer were loaded for electrophoresis at an appropriate voltage for 3 hours. The gel was transferred to a polyvinylidene difluoride membrane (Solarbio, Beijing, China) at 300 mA for complete protein transfer. The membrane was blocked with 5% skim milk and then incubated with the primary antibody at 4 ° C overnight. After rinsing 3 times with PBST, the membrane was incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit / mouse IgG (Zhongshan Jinqiao, Beijing, China) at room temperature for 1 hour. After spreading SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, MA, USA) for a few seconds, the immunoreactive bands were visualized by the ECL Chemiluminescent System (Bio-Rad, CA, USA).

[0136] Flow cytometry

[0137] When the cells were cultivated at 80-90% in 6 cm culture dishes, they were harvested with a cutting enzyme (Sigma-Aldrich, Mo, USA), and incubated at room temperature for 15 minutes with 50 μM concentration of Cy5-dual targeting peptides, protected from light. After incubation, the cells were washed 3 times with ice-cold PBS and resuspended in 200 μL PBS. Flow cytometry was performed on a CytoFLEX LX flow cytometer (Beckman Coulter, IN, USA), and 10,000 gated cells were finally collected for analysis. FlowJo software (v7.6, BD Biosciences, OR, USA) was finally used to analyze the results by flow cytometry.

[0138] Immunofluorescence staining

[0139] Approximately 5 × 104 cells were seeded onto fibronectin-coated coverslips in 24-well plates and fixed with 4% paraformaldehyde until the cells reached approximately 50% confluence. After permeabilization with 0.5% TritonX-100 (Millipore, MA, USA) for 10 min, the cells were blocked with 5% BSA for 0.5 h and subsequently incubated with Cy5-coupled peptide at a concentration of 80 μM at 4 °C overnight. The next day, after washing with phosphate-buffered saline (PBS), 1 μg / mL diaminophenylamine (DAPI) was added to stain the nuclei. Then, the coverslips were mounted with the Prolong Gold antifade technique (Invitrogen P26930, CA, USA) and visualized using a confocal laser scanning microscope (Zeiss LSM880, Oberkochen, Germany).

[0140] Immunohistochemistry

[0141] The samples were fixed with formalin, embedded in paraffin, and then cut into 5 μm sections. The sections were stripped in xylene and hydrated in gradient concentrations of ethanol and distilled water. Antigen retrieval was performed under high pressure with boiling citrate buffer (pH 6.0, MaxVision, Fujian, China). After blocking with 5% goat serum for 30 min, all samples were incubated with anti-igf2bp2 antibody (11601-1-AP, Protein Protein, IL, USA) at 4 °C overnight. Subsequently, the sections were washed three times with PBST and incubated with HRP-conjugated rabbit anti-rabbit antibody (MaxVision, Fujian, China) for half an hour at room temperature. The color reaction was performed with diaminobenzidine (DAB) (Maxion, Fujian, China) and then stained with hematoxylin (Zhongshan Jinqiao, Beijing, China). Visualization was performed using a slide scanning system (3DHistech Co., Ltd., Budapest, Hungary), and the images were captured and analyzed by a panoramic viewer (3DHistech Co., Ltd., Budapest, Hungary). The staining intensity was assessed using the software-automated quantitative histochemical score (h-score), and the expression was classified as negative (h-score <50), weak (50-99), moderate (100-199), or positive (200-300).

[0142] Near-infrared fluorescence imaging

[0143] Mice bearing subcutaneous esophageal tumors were injected with the equivalent of 50 μg of Cy5-dual targeting peptide via the tail vein. After cycling, mice were anesthetized with isoflurane and fluorescence images were captured by IVIS Spectrum (IVIS, MA, USA) at 10 min, 1 h, 2 h, 4 h, and 6 h with an excitation wavelength of 640 nm and an emission wavelength of 670 nm. Regions of interest (ROIs) were manually drawn to calculate the fluorescence intensity as the mean fluorescence intensity (MFI). In addition, tumors and major organs (bladder, liver, heart, lung, stomach, spleen, skin, kidney, colon, muscle, bone, and blood) were dissected and imaged for biodistribution analysis.

[0144] Data Analysis and Statistics

[0145] Statistical analysis was performed using GraphPad Prism software (v9.0, GraphPad, CA, USA). All data are presented as mean ± standard deviation (SD). Statistical significance between the two groups was determined by two-tailed Student's t-test or one-way analysis of variance (ANOVA). The threshold for statistical significance was set at p < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001).

[0146] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A targeted probe for ESCC imaging, characterized in that: The targeting probe is a Cy5-dual targeting peptide, and its polypeptide sequence is shown in SEQ ID NO:

1.

2. The targeted probe for ESCC imaging according to claim 1, characterized in that: The preparation of the targeting probe comprises the following steps: S1: Solvent treatment: DMF and methanol were soaked in G3 molecular sieves overnight before use to remove impurities and water; S2: Full swelling of the resin: weigh 2.0 g of blank Wang resin into a clean and dry reaction tube, add 15 mL of DMF, and activate at room temperature for 30 min; S3: Connect the first amino group: At room temperature, filter out the solvent from the previous step through a sand core, add 1mmol of a 5-fold molar excess of the first amino acid at the C-terminus, a 5-fold molar excess of DMAP, a 5-fold molar excess of DIC, and DMF as a solvent, and react at room temperature for 3 hours; after the reaction, wash with DMF 4-6 times, 5-6mL each time; then add an appropriate amount of pyridine and acetic anhydride in a volume ratio of 1:1, and react for 30 minutes; after the reaction, wash with DMF 4-6 times, 5-6mL each time; S4: Removal of Fmoc protecting group: remove the solvent from the previous step by suction, add 10 mL of 20% piperidine DMF solution to the resin, stir under N2 for 10 min, filter out the solution, add 10 mL of 20% piperidine DMF solution, stir under N2 for 5 min, filter out the solution, repeat this operation twice, wash with DMF 4 times and methanol 2 times, 5-6 mL each time; S5: Ninhydrin test for removal effect: Take out a small amount of resin, wash it three times with methanol, add one drop of ninhydrin, KCN, and phenol solution, heat at 105-110℃ for 5 minutes, and turn dark blue for positive reaction, indicating that the removal is complete, and the next step can be carried out; if it is colorless, it means that the protecting group is not completely removed, and the above deprotection operation needs to be repeated; S6: Removal of the second amino acid and Fmoc protecting group: weigh a 3-fold molar excess of the second amino acid at the C-terminus, a 3-fold molar excess of HBTU and a 3-fold molar excess of HOBT in a reaction tube, add an appropriate amount of DMF solution to completely dissolve it, then add a 10-fold molar excess of DIEA, react at room temperature for 40 minutes, wash with DMF 4-6 times, 5-6 mL each time; take a small amount of resin and detect it with ninhydrin detection reagent, it is colorless, then add 10 mL of 20% piperidine DMF solution to remove Fmoc, and perform it twice, 10 minutes and 5 minutes respectively, then wash it with DMF 4 times and methanol 2 times, 5-6 mL each time; take out a small amount of resin and detect it with ninhydrin detection reagent, and detect it as blue, then proceed to the next step of reaction; S7: Repeat step S6 in this way until the last amino acid at the N-terminus is synthesized, remove the Fmoc protecting group, and then drain; S8: Resin shedding and pure product separation and detection: Finally, use trifluoroacetic acid cutting solution to cut for 2 hours, filter the reaction solution, and obtain trifluoroacetic acid solution of the peptide. Use nitrogen to blow dry the cleavage solution as much as possible, then precipitate with ether, centrifuge, and then wash with ether 3-5 times to obtain a white solid. After dissolving in pure water, desalt and purify by HPLC, freeze-dry and precipitate crystals to obtain Cy5-dual targeting peptide.

3. Use of the targeted probe according to claim 1 or 2 in ESCC imaging.

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