CN116217664A - Peptides, molecular probes and applications targeting mHSP90 - Google Patents

Abstract

The embodiment of the invention discloses a polypeptide targeting mHSP90, a molecular probe and application thereof; the polypeptide targeting mHSP90 is: (i) polypeptide P7; or (ii) an amino acid sequence obtained by ligating a tag at the N-terminus and/or C-terminus of the polypeptide P7; or (iii) a polypeptide having the same function obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence of (i) or (ii). Polypeptide targeting mHSP90, nucleic acid molecule encoding the polypeptide, biological material containing the nucleic acid molecule, molecular probe targeting mHSP90 can be used for preparing molecular imaging agent and preventive and therapeutic drug for expression related abnormal related diseases; low cost, small molecular weight, good biocompatibility, strong penetrability, no immunogenicity, higher blood clearance rate, simple preparation, strong superiority in tumor targeted drug delivery, cancer diagnosis and other aspects, and has good application prospect.

Description

Peptides, molecular probes and applications targeting mHSP90

technical field

The invention belongs to the technical field of biomedical detection, and specifically relates to mHSP90-targeted polypeptides, molecular probes and applications.

Background technique

Malignant tumors are currently a major disease that seriously endangers human life. In recent decades, the global tumor morbidity and mortality are still showing a rapid upward trend. In recent years, advances in treatment technologies such as surgery, chemotherapy, radiation, targeted and immunotherapy have greatly improved the efficacy of tumor treatment, but the vast majority of cancer patients are still unable to obtain a radical cure, and are prone to recurrence and metastasis. The survival time and Quality of life is consistently low. Many tumors progress insidiously, and the patients are already at an advanced stage at the first diagnosis.

Heat shock protein 90 (Hsp90) is an important molecular chaperone protein in cells, which regulates the conformation, maturation and stability of more than 400 client proteins. Inhibition of Hsp90 can affect multiple signaling pathways in cells. In tumor cells, the expression level of Hsp90 is 2-10 times that of normal cells. Inhibiting the expression level of Hsp90 can trigger the degradation of client proteins and promote the killing of cells. Therefore, Hsp90 has become an important target for the treatment of various cancer types. Although various Hsp90 inhibitors have entered the clinical research stage one after another, no drug has yet been marketed. The main reason is that Hsp90 inhibitors cannot accurately target tumor cells and have potential toxic side effects.

Hsp90 is not only highly expressed in various tumor cells such as lung cancer, pancreatic cancer, and breast cancer, but also expressed on the tumor cell membrane. Scholars including our research group have confirmed the presence of cell membrane-bound Hsp90 in more than a dozen tumors, including lung cancer, liver cancer, pancreatic cancer, gastric cancer, liver cancer, cholangiocarcinoma, breast cancer, glioma, melanoma, and colorectal cancer. (Membrane-bound Hsp90, mHsp90), which is basically absent in normal cell membranes. mHsp90 localizes on the tumor cell membrane by interacting with lipid rafts and unsaturated phospholipids, regulates the functions of HER2, MMP2 and other proteins, and participates in the invasion and metastasis of tumor cells. It is expected to become an ideal molecular target for the treatment of human malignant tumors.

Molecular imaging is playing an increasingly important role in immunotherapy and personalized medicine. The preparation of molecular probes is the key to molecular imaging. However, the further application of antibody drugs is limited due to cumbersome preparation, poor in vitro stability, large molecules, difficult labeling, weak penetration, post-translational modification, and high cost.

In addition, chemotherapy drugs are more commonly used in cancer treatment. However, because targeted therapy has not been realized, the damage to normal cells is relatively large, so the dosage of the drug is greatly limited, and the patient's drug resistance and therapeutic effect are not very satisfactory.

Contents of the invention

In view of this, in order to solve the deficiencies in the prior art, the present invention provides a tumor-targeting conjugated drug targeting mHsp90 and its application strategy.

In one aspect, some embodiments disclose a polypeptide targeting mHSP90, the polypeptide being:

(i), polypeptide P7; or

(ii), the amino acid sequence obtained by attaching a tag to the N-terminal and/or C-terminal of the polypeptide P7; or

A polypeptide having the same function obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence of (iii), (i) or (ii).

In another aspect, some embodiments disclose nucleic acid molecules that encode polypeptides that target mHSP90.

In another aspect, some embodiments disclose biological materials containing nucleic acid molecules targeting mHSP90 polypeptides, including recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria or transgenic cell lines.

On the other hand, some embodiments disclose molecular probes targeting mHSP90, which are formed by linking signal units with polypeptides targeting mHSP90, and the signal units include radioisotopes, nuclear magnetic resonance contrast agents, fluorescent dyes, quantum dots, magnetic nanoparticles , superparamagnetic material, and at least one of ultrasonic microbubbles.

For the molecular probes targeting mHSP90 disclosed in some embodiments, the polypeptide and the signaling unit are connected through a linker molecule or a chelating agent;

Wherein, the linking molecule includes thioether, maleimido caproyl, disulfide bond, hydrazone bond, valine-citrulline dipeptide bond, amide, 6-tert-butoxycarbazinyl nicotinic acid, or 1 - Ethyl-3-(3-dimethylaminopropyl)-carbodiimide or N-hydroxysuccinimide;

Chelating agents include DTPA, DOTA, NOTA or TETA.

Further, some embodiments disclose the application of mHSP90-targeting polypeptides, nucleic acid molecules, biological materials, or molecular probes targeting mHSP90, including:

(1) For preparing medicines for diseases related to abnormal expression of mHSP90;

(2) It is used to prepare molecular imaging agents for diseases related to abnormal expression of mHSP90.

For the application disclosed in some embodiments, the drug includes a polypeptide targeting mHSP, a preparation for killing cancer cells coupled with a polypeptide targeting mHSP, and an optional drug carrier; the preparation for killing cancer cells is a preparation for killing cancer cells Chemopharmaceuticals, biopharmaceuticals, nanomedicines, radiopharmaceuticals, photothermal therapy or photodynamic therapy drugs for cancer cells.

For the applications disclosed in some embodiments, the diseases related to abnormal expression of mHSP90 are tumors, including pancreatic cancer, lung cancer, neurotumor, breast cancer, gastric cancer, colorectal cancer, liver cancer, osteosarcoma, cholangiocarcinoma, thyroid cancer, angiosarcoma, gastric cancer, ovarian cancer cancer, cervical cancer or prostate cancer.

In the application disclosed in some embodiments, the molecular imaging agent is used in fluorescence imaging, positron emission tomography, single photon emission tomography, magnetic resonance imaging, photoacoustic imaging, ultrasound imaging or other in vivo imaging fusion imaging techniques.

For the applications disclosed in some embodiments, the diseases related to abnormal expression of mHSP90 are tumors, including pancreatic cancer, lung cancer, neurotumor, breast cancer, gastric cancer, colorectal cancer, liver cancer, osteosarcoma, cholangiocarcinoma, thyroid cancer, angiosarcoma, gastric cancer, ovarian cancer cancer, cervical cancer or prostate cancer.

The drugs disclosed in some embodiments include mHSP90-targeted polypeptides, nucleic acid molecules, biological materials, or mHSP90-targeted molecular probes.

Unlike existing tumor targeted therapy and imaging targets such as epidermal growth factor receptor, integrin, and folic acid receptor, mHsp90 basically does not exist on the cell membrane of normal cells. mHsp90 is a specific tumor-specific antigen, and P7 polypeptide can specifically By using this characteristic, it can be connected with imaging units such as radioactive isotopes, magnetic resonance contrast agents, fluorescent dyes, quantum dots, magnetic nanoparticles, superparamagnetic materials, and ultrasonic microbubbles to construct molecular targeting probes. Tumor diagnosis, treatment and prognosis evaluation provide a new way.

The mHSP90-targeting polypeptides disclosed in the embodiments of the present invention can highly express cell membrane-bound HSP90, and the mHSP90-targeting polypeptides, nucleic acid molecules encoding the polypeptides, biological materials containing the nucleic acid molecules, and mHSP90-targeting molecular probes can Used to prepare detection reagents and molecular imaging agents for mHSP90 expression-related abnormalities and related diseases; low cost, small molecular weight, good biocompatibility, strong penetrability, non-immunogenicity, and faster blood clearance rate, preparation Simple, tumor-targeted administration in cancers including pancreatic cancer, lung cancer, neuro-oncology, breast cancer, gastric cancer, colorectal cancer, liver cancer, osteosarcoma, cholangiocarcinoma, thyroid cancer, angiosarcoma, gastric cancer, ovarian cancer, cervical cancer or prostate cancer It has great advantages in medicine and cancer diagnosis, and has a good application prospect.

Description of drawings

The FITC-P7 chromatographic analysis figure that Fig. 1 embodiment 1 discloses;

The FITC-P7 mass spectrometry figure that Fig. 2 embodiment 1 discloses;

The ICG-P7 mass spectrometry figure that Fig. 3 embodiment 2 discloses;

The ICG-P7 in vivo imaging results figure disclosed in Fig. 4 embodiment 2;

The fluorescent probe spectrum analysis diagram disclosed in Fig. 5 embodiment 3;

Fig. 6 The uptake and blocking effect of fluorescent probes by tumor cells disclosed in Examples 4, 5, and 6;

The DOTA-P7 structural formula for NMR detection disclosed in Figure 7 embodiment 7;

Amino acid sequence of polypeptide P7: L(Leu)P(Pro)L(Leu)T(Thr)P(Pro)L(Leu)P(Pro).

Detailed ways

The word "embodiment" is used here exclusively, and any embodiment described as "exemplary" is not necessarily to be construed as superior or better than other embodiments. In the performance index tests in the embodiments of the present invention, unless otherwise specified, conventional test methods in the art are used. It should be understood that the terms described in the embodiments of the present invention are only used to describe specific implementation manners, and are not used to limit the content disclosed in the embodiments of the present invention.

Unless otherwise specified, the technical and scientific terms used herein have the same meanings commonly understood by those of ordinary skill in the technical field to which the embodiments of the present invention belong; as other unspecified test methods and technical means in the embodiments of the present invention all refer to Experimental methods and technical means commonly used by those skilled in the art.

The terms "substantially" and "approximately" are used herein to describe small fluctuations. For example, they may refer to less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ± 0.1%, if less than or equal to ±0.05%. Numerical data expressed or presented herein in a range format are used for convenience and brevity only, and should therefore be construed flexibly to include not only the values expressly recited as the boundaries of that range, but also all individual values or values contained within that range. subrange. For example, a numerical range of "1 to 5%" should be interpreted to include not only the explicitly recited value of 1% to 5%, but also include individual values and subranges within the indicated range. Accordingly, individual values such as 2%, 3.5% and 4%, and subranges such as 1% to 3%, 2% to 4% and 3% to 5% etc. are included in this numerical range. The same principle applies to ranges reciting only one numerical value. Moreover, such an interpretation applies regardless of the breadth of the range or the characteristics described.

Herein, including in the claims, conjunctions such as "comprises," "comprises," "with," "has," "containing," "relates to," "contains," etc., are to be construed as open-ended , which means "including but not limited to". Only the conjunctions "consisting of" and "consisting of" are closed conjunctions.

In order to better illustrate the contents of the present invention, numerous specific details are given in the following specific examples. It will be understood by those skilled in the art that the present invention may be practiced without certain of the specific details. In the embodiments, some methods, means, instruments, equipment, etc. that are well known to those skilled in the art are not described in detail, so as to highlight the gist of the present invention.

On the premise of no conflict, the technical features disclosed in the embodiments of the present invention can be combined arbitrarily, and the obtained technical solutions belong to the content disclosed in the embodiments of the present invention.

In some embodiments, the polypeptide targeting mHSP90 is:

(i), polypeptide P7; or,

(ii), the amino acid sequence obtained by attaching a tag to the N-terminal and/or C-terminal of the polypeptide P7; or,

A polypeptide having the same function obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence of (iii), (i) or (ii).

Generally, mHSP90 is tumor cell membrane-bound HSP90, and the polypeptide targeting mHSP90 is polypeptide P7, whose amino acid sequence is LPLTPLP, and has high specific affinity for mHSP90. mHSP90 can localize on the tumor cell membrane by interacting with lipid rafts and unsaturated phospholipids, regulate the functions of HER2, MMP2 and other proteins, participate in the invasion and metastasis of tumor cells, etc., and can be used as an ideal molecular target for the treatment of human malignant tumors.

Further, a polypeptide comprising the amino acid sequence obtained after the N-terminal and/or C-terminal of the polypeptide P7 is linked to a tag, or a polypeptide having the same function obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence of the polypeptide P7, or comprising A polypeptide with the same function obtained by substituting, deleting and/or adding one or more amino acid sequences to the amino acid sequence obtained after the N-terminal and/or C-terminal of the polypeptide P7 is linked with a tag, which can be an ideal molecular target for the treatment of human malignant tumors Affinity ligand for mHSP90.

In some embodiments, the polypeptide P7 is a P7 polypeptide synthesized by using solid-phase synthesis technology and Fmoc synthesis strategy, and has a high specific affinity for mHSP 90.

Some embodiments disclose nucleic acid molecules encoding mHSP90-targeting polypeptides.

Some embodiments disclose biological materials containing nucleic acid molecules encoding mHSP90-targeting polypeptides, including recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

The DNA fragment disclosed in some embodiments, the DNA fragment comprises the amino acid sequence encoding the polypeptide P7.

Some embodiments disclose the use of polypeptides targeting mHSP90, including:

(1) Drugs for preparing diseases related to abnormal expression of mHSP90; usually drugs are used to prevent or treat diseases related to abnormal expression of mHSP90;

(2) It is used to prepare molecular imaging agents for diseases related to abnormal expression of mHSP90.

In some embodiments, the polypeptide targeting mHSP90 is used to prepare a drug for the prevention or treatment of diseases related to abnormal expression of mHSP, and the drug includes a polypeptide targeting mHSP, a preparation for killing cancer cells coupled with a polypeptide targeting mHSP, And an optional drug carrier; the preparation for killing cancer cells is a chemical drug, biological drug, nano drug, radiopharmaceutical, photothermal therapy or photodynamic therapy drug for killing cancer cells.

In some more preferred embodiments, the preparations used to kill cancer cells are alkylating agents, anti-metabolite drugs, anti-tumor natural drugs, anti-tumor antibiotics, anti-tumor neovascularization drugs, metal complexes or tumor radiation targeting markers .

In some embodiments, the polypeptide targeting mHSP90 is used to prepare a molecular imaging agent for diseases related to abnormal expression of mHSP, and the molecular imaging agent is used for fluorescence imaging, positron emission tomography, single photon emission tomography, magnetic resonance imaging, Photoacoustic imaging, ultrasound imaging or other fusion imaging techniques of in vivo imaging.

The application of the polypeptide targeting mHSP90 disclosed in some embodiments, the abnormal expression of mHSP90 related diseases are tumors, including pancreatic cancer, lung cancer, neurotumor, breast cancer, gastric cancer, colorectal cancer, liver cancer, osteosarcoma, cholangiocarcinoma, thyroid cancer, Angiosarcoma, stomach, ovary, cervix, or prostate cancer.

Some embodiments disclose molecular probes targeting mHSP90, which are formed by linking signal units targeting mHSP90 polypeptides, the signal units include radioisotopes, nuclear magnetic resonance contrast agents, fluorescent dyes, quantum dots, magnetic nanoparticles, superparamagnetic At least one of materials and ultrasonic microbubbles.

For the mHSP90-targeting molecular probes disclosed in some embodiments, the mHSP90-targeting polypeptide and the signaling unit are connected through a linker molecule, and the connection method includes mixing with a polymer, or non-covalent bond connection, or using other chemical reactions to connect The signal unit is directly linked to the polypeptide; wherein, the linking molecule includes thioether, maleimido caproyl, disulfide bond, hydrazone bond, valine-citrulline dipeptide bond, amide, 6-tert-butoxycarbonyl Nicotinic acid, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, or N-hydroxysuccinimide; for example, 6-tert-butoxycarbonyl nicotinic acid with peptide The Pro carboxyl group at the C-terminal reacts; 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide or N-hydroxysuccinimide reacts with the carboxyl group at the C-terminal of the polypeptide; or the long-chain diacid reacts with Pro valine-citrulline dipeptide bonds and disulfide bonds are introduced at both ends of the P7 polypeptide; the polymer is polyethylene glycol (PEG), polyvinyl alcohol (PVA), cyclodextrin, polyamide - Any one or a combination of at least two of amine dendrimers (PAMAM), polylactic acid (PLA), polylactic acid-ethanolamine (PLGA).

For the molecular probe targeting mHSP90 disclosed in some embodiments, the polypeptide targeting mHSP90 is connected to the signal unit through a chelating agent, and the chelating agent includes DTPA, DOTA, NOTA or TETA; usually, the amino group at the N-terminal of the polypeptide reacts with the carboxyl group of the chelating agent Perform coupling.

The molecular probes targeting mHSP90 disclosed in some embodiments are used to prepare molecular imaging agents for diseases related to abnormal expression of mHSP90.

In some embodiments, the conjugates of the polypeptide targeting mHSP90 and the chelating agents DTPA and DOTA can chelate paramagnetic Gd for MRI imaging; the conjugates of the polypeptide and the chelating agents DTPA, DOTA, NOTA or TETA can be ^chelated68 Ga or ⁶⁴ Cu for PET imaging.

The molecular probes targeting mHSP90 disclosed in some embodiments are used to prepare reagents for diagnosing diseases related to abnormal expression of mHSP90, and to be used as drugs or imaging preparations for immunotherapy, prevention or diagnosis of cancer.

In some embodiments, the targeted molecular probe targeting tumor cell membrane-bound mHSP90 includes a signaling unit, a targeting affinity unit, and a linker for connecting the signaling unit and the targeting affinity unit, wherein the signaling unit is The part that can be detected by imaging equipment, the targeting affinity unit is a polypeptide part that specifically binds to mHSP90.

In some embodiments, the molecular probes targeting mHSP90 can be used for fluorescence imaging, positron emission tomography, single photon emission tomography, magnetic resonance imaging, photoacoustic imaging, ultrasound imaging or other in vivo imaging fusion in patients imaging technology.

In some preferred embodiments, the molecular probes targeting mHSP90 can be used to perform fluorescence imaging, PET/CT or PET/MRI on patients.

The application of molecular probes targeting mHSP90 disclosed in some embodiments, the abnormal expression of mHSP90 related diseases are tumors, including pancreatic cancer, lung cancer, neurotumor, breast cancer, gastric cancer, colorectal cancer, liver cancer, osteosarcoma, cholangiocarcinoma, thyroid cancer carcinoma, angiosarcoma, stomach, ovary, cervix, or prostate.

The technical details are further exemplified below in conjunction with the embodiments.

Example 1

Preparation and fluorescence detection of FITC-P7 polypeptide fluorescent molecular probe

The preparation and fluorescence detection of FITC-P7 polypeptide fluorescent molecular probe specifically include:

Weigh 100 mg of wang-Cys resin, and cycle sequentially according to the sequence of the P7 polypeptide according to the solid-phase peptide synthesis strategy;

After the coupling reaction, deprotect and wash;

Add ε-aminocaproic acid to react at room temperature for 2 hours. After deprotection, fluorescein isothiocyanate ( FITC) was mixed with 50 mg resin and reacted overnight at room temperature in the dark, and the molar ratio of FITC to resin was 1:1;

After the reaction is over, isopropanol cleans the methanol for dehydration;

Lyse the liquid into the above resin, remove the side chain protecting group, and dry it in vacuum to obtain the crude polypeptide FITC fluorescent conjugate;

MALDI-TOF identification and HPLC purification were used for subsequent experiments.

The results are shown in Figure 1 and Figure 2, and the results showed that FITC-P7 with a purity of more than 95% was obtained.

Example 2

Preparation and functional verification of ICG-P7, Cy-5, Cy-7 polypeptide imaging preparations

Example 2 takes the preparation of ICG-P7 polypeptide fluorescent conjugate as an example to demonstrate the preparation and functional verification of the P7 polypeptide near-infrared in vivo imaging preparation. Specifically include:

Weigh 1 mg of the synthetic P7 polypeptide prepared in Example 1, dissolve it in 1×PBS, weigh 0.5 mg of indocyanine green-maleimide (ICG-MAL) and dissolve it in 500 μL deionized water, and mix the two , adjust the pH to about 7.4, shake the reaction at room temperature for 24 hours, and perform mass spectrometry detection and HPLC purification after freeze-drying;

The obtained product was lyophilized after dialysis to obtain an ICG-polypeptide imaging preparation for mouse fluorescence imaging.

Pancreatic cancer cell line PANC-1 was cultured in DMEM medium containing 10% fetal bovine serum, and injected subcutaneously into the right hind limb of Balb/c nude mice at 1×106 until the tumor grew to 50 mm ³ ;

Weigh 1 mg of ICG-P7 polypeptide and dissolve it in 1 mL of 1×PBS, inject 150 μL of ICG-peptide into the tail vein for half an hour, use the IVIS Spectrum small animal in vivo optical three-dimensional imaging system to collect signals;

After 24 hours of dissection, the main organs were taken for in vitro fluorescence distribution imaging.

The results are shown in Figure 3. It was identified by MALDI-TOF-MS that ICG was successfully labeled on the P7 polypeptide.

The polypeptide imaging preparation prepared in Example 2 above was injected into the tumor-bearing mice through the tail vein, as shown in Figure 4, at the time point of 5 hours, ICG-P7 was significantly enriched in the tumor site, while ICG only A small amount was enriched in the tumor site. At the time point of 24h and 48h, the fluorescence in the tumor site of the mice in the ICG-P7 group gradually weakened, while no fluorescence was seen in the tumor site of the mice in the ICG group, indicating that ICG-P7 has a good effect on tumors in vivo. Targeted, and can be metabolized and excreted over time.

Example 3

Fluorescent Probe Absorption and Emission Spectrum Detection

Specific methods for absorption wavelength detection include:

Dilute the Cy5/Cy5-P7 stock solution to a concentration of 5 μM using PBS;

Cy5 and Cy5-P7 were added to a transparent bottom 96-well plate at 200 μl/well, three parallel wells were placed in the dark;

Turn on the fluorescent microplate reader, select the absorption spectrum scan, set the parameters to the detection range of 400nm-700nm, and the detection step to 1nm;

Click the degree to read the absorbance value of each well.

Emission spectroscopy detection methods include:

Dilute the Cy5-P7 stock solution to a concentration of 5 μM with PBS;

Cy5 and Cy5-P7 were added to a black 96-well plate at 200 μl/well, three parallel wells were placed in the dark;

Turn on the fluorescent microplate reader, select the emission spectrum scan, set the parameters as the excitation wavelength is 600nm, the detection range is 620nm-700nm, and the detection step is 1nm;

Click the degree to read the fluorescence value of each well.

The results showed that the absorption spectrum and emission spectrum of the fluorescent probe did not change significantly after coupling P7, indicating that the fluorescence properties of the fluorescent probe did not change after coupling P7, as shown in FIG. 5 .

Example 4

Flow Cytometry Detection of Molecular Probe Uptake by Tumor Cells

Specific methods include:

Adjust the cell concentration to 2×105/ml;

Add 1ml/well of cell suspension into the six-well plate, make up to 2ml with culture medium, and culture in the incubator overnight;

The next day, the culture medium was discarded, and 2ml of serum-free DMEM high-glucose medium containing 10nM Cy5/Cy5-P7 was added, at 37°C for 1h;

Discard the culture medium containing the fluorescent probe, wash with PBS 3 times, add EDTA-free trypsin to digest for 4 minutes, pipette the cells and transfer them to a 1.5ml centrifuge tube, centrifuge at 600g for 5 minutes, and gently aspirate and discard the supernatant;

Add 500μl PBS to mix by pipetting, centrifuge at 600g for 5min, and discard the supernatant;

Add 500 μl PBS to resuspend the cells, and perform flow cytometry detection on the machine.

The results showed that the PANC-1 cells were incubated with the same molar concentration of fluorescent dyes, and the final average fluorescence intensity in the cells was detected by flow cytometry. The average fluorescence intensity of the cells incubated with Cy5 was similar to that of the control group, while the average fluorescence intensity of the cells incubated with Cy5-P7 The fluorescence intensity was significantly enhanced, as shown in Figure 6.

Example 5

Study on the Blocking of Molecular Probe Uptake by Tumor Cells

Specific methods include:

Discard the original culture medium for the cells, add 2ml fresh culture medium, add 20μl Hsp90 monoclonal antibody AC88 to the AC88 antibody blocking group, the final concentration is 10μg/ml, and incubate at 4°C for 30min;

Keep the original culture solution, add Cy5/Cy5-P7 solution to each well according to grouping, the final concentration is 10nM, 37°C, 1h;

Use Hsp90 monoclonal antibody to block the uptake process of Cy5-P7, and detect it by flow cytometry and fluorescent staining respectively;

The results showed that the average fluorescence intensity in the cells decreased significantly after blocking, as shown in FIG. 6 .

Example 6

Detection of Cy5-P7 Uptake by PANC-1 Cells by Immunofluorescence Staining

Specific methods include:

Prepare PANC-1 cells into 2×105/ml cell suspension;

Put an autoclaved cover glass in a six-well plate to make cell slides, add 1ml of cell suspension per well, make up to 2ml with culture medium, and culture in an incubator overnight;

Discard the original culture medium, wash twice with PBS, and add 2ml of serum-free DMEM high-glucose medium;

Add 20 μl of Hsp90 monoclonal antibody AC88 to the AC88 antibody blocking group with a final concentration of 10 μg/ml, and incubate at 4°C for 30 minutes; keep the original culture medium, and add Cy5/Cy5-P7 liquid to each well according to grouping, with a final concentration of 10 μM, and incubate at 37°C , 1h;

Discard the culture medium, wash with PBS for 3 minutes, twice, add 4% paraformaldehyde and incubate at room temperature for 15 minutes;

Discard paraformaldehyde, wash with PBS for 3 minutes, twice, add DAPI staining solution, and protect from light at room temperature for 30 minutes;

Discard the DAPI dye solution, wash with ultrapure water for 3 minutes, twice;

Wipe off the excess liquid, seal the slides with anti-fluorescence quenching agent, observe with a confocal microscope immediately or store at 4°C.

The results are shown in Figure 6: the average fluorescence intensity of the cells incubated with Cy5-P7 was significantly enhanced.

Example 7

Synthetic method of DOTA-P7 for nuclear magnetic detection

Using 2-CTC resin as the starting resin, the Fmoc solid phase method is used to gradually couple amino acids Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Thr-OH, Fmoc-Leu-OH, Fmoc -Pro-OH, Fmoc-Leu-O, use TUBU/HOBt/DIEA as the amino acid condensing agent, after the amino acid coupling is completed, remove Fmoc, use TristBu-DOTA (1,4,7,10-tetraazacyclodeca Tri-tert-butyl alkane-1,4,7,10-tetraacetate)/TBTU/HOBt/DIEA capped. After each step of the above reaction, the reaction is controlled by K test/C test. If the condensation reaction of a certain amino acid is not complete, repeat the condensation once. After coupling, wash and dry.

Cleavage the peptide resin with TFA lysate and remove the side chain protecting group, control the temperature at 20-30°C, settle with isopropyl ether, and dry to obtain the crude DOTA-P7 product, which will be further purified in one step and converted to salt in two steps to obtain the finished product of DOTA-P7. The structural formula is shown in Figure 7.

The mHSP90-targeting polypeptide disclosed in the embodiments of the present invention can highly express cell membrane-bound HSP90, and can be used to prepare detection reagents for mHSP90 expression-related abnormalities and related diseases and prepare molecular imaging agents for mHSP90 expression-related abnormalities-related diseases; targeting The molecular probe of mHSP90 can be used to prepare molecular imaging agents for diseases related to abnormal expression of mHSP90, with low cost, small molecular weight, good biocompatibility, strong penetrability, non-immunogenicity, and faster blood clearance rate , simple preparation, etc., in the treatment of pancreatic cancer, lung cancer, neurotumor, breast cancer, gastric cancer, colorectal cancer, liver cancer, osteosarcoma, bile duct cancer, thyroid cancer, angiosarcoma, gastric cancer, ovarian cancer, cervical cancer or prostate cancer It has strong advantages in tumor-targeted drug delivery and cancer diagnosis, and has a good application prospect.

The technical solutions disclosed in the embodiments of the present invention and the technical details disclosed in the embodiments are only illustrative of the inventive concept of the present invention, and do not constitute a limitation to the technical solutions of the embodiments of the present invention. All conventional changes, substitutions or combinations, etc., have the same inventive concept as the present invention, and are within the protection scope of the claims of the present invention.

Claims (10)

1. A polypeptide targeting mHSP90, characterized in that the polypeptide is:

(i) Polypeptide P7; or (b)

(ii) An amino acid sequence obtained by connecting a tag to the N-terminal and/or C-terminal of the polypeptide P7; or (b)

(iii) A polypeptide having the same function obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence of (i) or (ii).

2. A nucleic acid molecule encoding the mHSP 90-targeting polypeptide of claim 1.

3. A biological material comprising the nucleic acid molecule of claim 2, comprising a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, an engineering bacterium, or a transgenic cell line.

4. A molecular probe targeting mHSP90, comprising the mHSP 90-targeting polypeptide of claim 1 and a signaling unit comprising at least one of a radioisotope, a nuclear magnetic resonance contrast agent, a fluorescent dye, a quantum dot, a magnetic nanoparticle, a superparamagnetic material, an ultrasound microbubble.

5. The mHSP90 targeting molecular probe of claim 4 wherein the mHSP90 targeting polypeptide is linked to the signal unit by a linker molecule or by a chelator;

wherein the linker molecule comprises thioether, maleimidocaproyl, disulfide, hydrazone, valine-citrulline dipeptide, amide, 6-t-butoxycarbonyl hydrazinonicotinic acid, or 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide or N-hydroxysuccinimide;

the chelating agent comprises DTPA, DOTA, NOTA or TETA.

6. Use of the mHSP90 targeting polypeptide of claim 1, the nucleic acid molecule of claim 2, the biological material of claim 3, or the mHSP targeting molecular probe of claim 4 or 5, comprising:

(1) For the preparation of a medicament for the treatment or prevention of a disease associated with abnormal mHSP90 expression;

(2) A molecular imaging agent for preparing a disease related to abnormal mHSP90 expression.

7. The use of claim 6, wherein the medicament comprises the mHSP 90-targeting polypeptide of claim 1, a formulation for killing cancer cells coupled to the mHSP 90-targeting polypeptide, and optionally a pharmaceutical carrier;

the preparation for killing cancer cells is a chemical drug, a biological drug, a nano drug, a radioactive drug, a photo-thermal treatment or a photodynamic treatment drug for killing cancer cells.

8. The use according to claim 6, wherein the disease associated with abnormal expression of mHSP90 is a tumor comprising pancreatic cancer, lung cancer, neurological tumor, breast cancer, gastric cancer, colorectal cancer, liver cancer, osteosarcoma, cholangiocarcinoma, thyroid cancer, angiosarcoma, gastric cancer, ovarian cancer, cervical cancer or prostate cancer.

9. The use according to claim 6, wherein the molecular imaging agent is used in fluorescence imaging, positron emission tomography, single photon emission tomography, magnetic resonance imaging, photoacoustic imaging, ultrasound imaging or other fused imaging techniques in vivo imaging.

10. A medicament comprising a polypeptide targeting msp 90 according to claim 1, a nucleic acid molecule according to claim 2, a biological material according to claim 3, or a molecular probe targeting msp 90 according to claim 4 or 5.

Images