CN116217664A - Polypeptide targeting mHSP90, molecular probe and application - 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

Polypeptide targeting mHSP90, molecular probe and application

Technical Field

The invention belongs to the technical field of biomedical detection, and particularly relates to a polypeptide targeting mHSP90, a molecular probe and application thereof.

Background

Malignant tumors are a serious disease that is currently seriously harmful to human life, and the incidence and death rate of global tumors still show a rapid rising trend in recent decades. In recent years, the progress of treatment technologies such as surgery, chemotherapy, radiation, targeting, immunotherapy and the like greatly improves the tumor treatment effect, but most tumor patients still cannot be radically treated, relapse and metastasis are easy to occur, and the survival time and life quality of malignant tumor patients are always lower. Many tumors progress in hidden, and patients have advanced first diagnosis.

Heat shock protein 90 (Hsp 90) is an important molecular chaperone protein in cells, regulating conformation, maturation and stability of more than 400 client proteins, inhibiting Hsp90 can affect multiple signaling pathways in cells. In tumor cells, the expression level of Hsp90 is 2-10 times that of normal cells, and inhibition of the expression level of Hsp90 can trigger degradation of client proteins and promote killing of cells, so that Hsp90 becomes an important target for treating various cancer types. Although various Hsp90 inhibitors enter clinical research stages successively, no drugs are marketed at present, mainly because Hsp90 inhibitors cannot be precisely aimed at tumor cells and have potential toxic and side effects.

Hsp90 is not only highly expressed in various tumor cells such as lung cancer, pancreatic cancer, breast cancer and the like, but also expressed on tumor cell membranes. The scholars including the subject group have confirmed that cell Membrane-bound Hsp90 (mHsp 90) exists in more than ten kinds of tumors such as lung cancer, liver cancer, pancreatic cancer, stomach cancer, liver cancer, cholangiocarcinoma, breast cancer, glioma, melanoma, colorectal cancer, and the like, but is substantially absent in normal cell membranes. The mHsp90 is positioned on tumor cell membranes through interaction with lipid rafts and unsaturated phospholipids, regulates and controls protein functions such as HER2, MMP2 and the like, participates in tumor cell invasion and metastasis processes and the like, and is expected to become an ideal molecular target for treating human malignant tumors.

Molecular imaging plays an increasingly important role in immunotherapy and personalized medicine. Wherein the preparation of molecular probes is critical for molecular imaging. However, antibody drugs have the problems of complicated preparation, poor in vitro stability, larger molecules, difficult labeling, weak penetrating power, post-translational modification, high cost and the like, so that the further application of the antibody drugs is limited.

In addition, chemotherapy drugs are currently more commonly used in cancer treatment. However, since the targeting treatment is not realized, the damage to normal cells is large, the dosage is greatly limited, and the drug resistance and the treatment effect of patients are not ideal.

Disclosure of Invention

In view of the above, in order to solve the defects in the prior art, the invention provides a tumor targeting coupling drug taking mHsp90 as a target point and an application strategy thereof.

In one aspect, some embodiments disclose a polypeptide that targets msp 90, which 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).

In another aspect, some embodiments disclose nucleic acid molecules for encoding polypeptides targeting mHSP 90.

In yet another aspect, some embodiments disclose biological materials comprising nucleic acid molecules that target a polypeptide of mHSP90, including recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineering bacteria, or transgenic cell lines.

In yet another aspect, some embodiments disclose a molecular probe targeting mHSP90, formed by linking a signal 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 to a polypeptide targeting mHSP 90.

Some embodiments disclose molecular probes targeting mHSP90 wherein the polypeptide is linked to the signaling unit by a linker molecule or by a chelator;

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

chelating agents include DTPA, DOTA, NOTA or TETA.

Further, some embodiments disclose the use of a polypeptide, nucleic acid molecule, biological material, or molecular probe that targets mHSP90, comprising:

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

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

Some embodiments disclose the use of a drug comprising a polypeptide that targets an mHSP, a formulation coupled to the polypeptide that targets an mHSP for killing cancer cells, and optionally a drug carrier; the preparation for killing cancer cells is chemical medicine, biological medicine, nanometer medicine, radioactive medicine, photothermal therapy or photodynamic therapy medicine for killing cancer cells.

For some embodiments, disclosed is an application, the disease associated with abnormal mHSP90 expression is a tumor, including pancreatic cancer, lung cancer, neural tumor, breast cancer, gastric cancer, colorectal cancer, liver cancer, osteosarcoma, cholangiocarcinoma, thyroid cancer, angiosarcoma, gastric cancer, ovarian cancer, cervical cancer, or prostate cancer.

Some embodiments disclose applications of molecular imaging agents for fluorescence imaging, positron emission tomography, single photon emission tomography, magnetic resonance imaging, photoacoustic imaging, ultrasound imaging, or other fused imaging techniques of living body imaging.

For some embodiments, disclosed is an application, the disease associated with abnormal mHSP90 expression is a tumor, including pancreatic cancer, lung cancer, neural tumor, breast cancer, gastric cancer, colorectal cancer, liver cancer, osteosarcoma, cholangiocarcinoma, thyroid cancer, angiosarcoma, gastric cancer, ovarian cancer, cervical cancer, or prostate cancer.

Some embodiments disclose a drug comprising a polypeptide, nucleic acid molecule, biological material that targets mHSP90, or a molecular probe that targets mHSP 90.

Unlike available tumor targeting treatment and imaging targets, such as EGF receptor, integrin and folic acid receptor, there is basically no mHsp90 in the cell membrane of normal cell, and mHsp90 is one specific tumor specific antigen, and P7 polypeptide can combine with mHsp90 specifically.

The polypeptide targeting the mHSP90 disclosed by the embodiment of the invention can be used for carrying out high expression on cell membrane-associated HSP90, the polypeptide targeting the mHSP90, a nucleic acid molecule for encoding the polypeptide, a biological material containing the nucleic acid molecule and a molecular probe targeting the mHSP90 can be used for preparing detection reagents and molecular imaging agents for related diseases of abnormal mHSP90 expression; low cost, small molecular weight, good biocompatibility, strong penetrability, no immunogenicity, rapid blood clearance rate and simple preparation, and has strong superiority in the aspects of tumor targeted drug delivery, cancer diagnosis and the like of pancreatic cancer, lung cancer, nerve tumor, breast cancer, gastric cancer, colorectal cancer, liver cancer, osteosarcoma, cholangiocarcinoma, thyroid cancer, angiosarcoma, gastric cancer, ovarian cancer, cervical cancer or prostate cancer, and has good application prospect.

Drawings

FIG. 1 shows a chart of FITC-P7 chromatography as disclosed in example 1;

FIG. 2 is a chart of FITC-P7 mass spectrometry as disclosed in example 1;

FIG. 3 is a chart of mass spectrometry of ICG-P7 disclosed in example 2;

FIG. 4 is a graph of the results of in vivo imaging of ICG-P7 as disclosed in example 2;

FIG. 5 is a spectroscopic analysis of the fluorescent probe disclosed in example 3;

FIG. 6 graphs of uptake and blocking effects of fluorescent probes by tumor cells as disclosed in examples 4, 5, and 6;

FIG. 7 example 7 discloses DOTA-P7 for nuclear magnetic resonance detection;

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

Detailed Description

The word "embodiment" as used herein does not necessarily mean that any embodiment described as "exemplary" is preferred or advantageous over other embodiments. Performance index testing in the examples of the present invention, unless otherwise specified, was performed using conventional testing methods in the art. It should be understood that the terminology used in the description of the embodiments of the invention presented is for the purpose of describing particular embodiments only, and is not intended to be limiting of the disclosure of the embodiments of the invention.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention belong; other test methods and techniques not specifically identified in the examples of the present invention are those generally employed by those skilled in the art.

The terms "substantially" and "about" 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%, such as less than or equal to ±0.05%. Numerical data presented or represented herein in a range format is used only for convenience and brevity and should therefore be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range. For example, a numerical range of "1 to 5%" should be interpreted to include not only the explicitly recited values of 1% to 5%, but also include individual values and sub-ranges within the indicated range. Thus, 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 being described.

In this document, including the claims, conjunctions such as "comprising," including, "" carrying, "" having, "" containing, "" involving, "" containing, "and the like are to be construed as open-ended, i.e., to mean" including, but not limited to. Only the conjunctions "consisting of … …" and "consisting of … …" are closed conjunctions.

Numerous specific details are set forth in the following examples in order to provide a better understanding of the present invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In the examples, some methods, means, instruments, devices, etc. well known to those skilled in the art are not described in detail in order to highlight the gist of the present invention.

On the premise of no conflict, the technical features disclosed by the embodiment of the invention can be combined at will, and the obtained technical scheme belongs to the disclosure of the embodiment of the invention.

In some embodiments, the polypeptide that targets mHSP90 is:

(i) Polypeptide P7; or alternatively, the first and second heat exchangers may be,

(ii) An amino acid sequence obtained by connecting a tag to the N-terminal and/or C-terminal of the polypeptide P7; or alternatively, the first and second heat exchangers may be,

(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).

Generally, the mHSP90 is tumor cell membrane-associated HSP90, the polypeptide targeting the mHSP90 is polypeptide P7, the amino acid sequence of the polypeptide is LPLTPLP, and the polypeptide has high specific affinity to the mHSP 90. The mHSP90 can be positioned on tumor cell membranes through interaction with lipid rafts and unsaturated phospholipids, regulate and control protein functions such as HER2, MMP2 and the like, participate in tumor cell invasion and metastasis processes and the like, and can be used as an ideal molecular target for treating human malignant tumors.

Further, a polypeptide comprising an amino acid sequence obtained by linking a tag to the N-terminus and/or the C-terminus of the polypeptide P7, 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 a polypeptide having the same function obtained by substituting, deleting and/or adding one or more amino acid sequences to the amino acid sequence obtained by linking a tag to the N-terminus and/or the C-terminus of the polypeptide P7, can be directed against an affinity ligand of an ideal molecular target mHSP90 for treating human malignant tumors.

In some embodiments, the polypeptide P7 is a P7 polypeptide synthesized using solid phase synthesis techniques using Fmoc synthesis strategies, with high specific affinity for mHSP 90.

Some embodiments disclose nucleic acid molecules encoding polypeptides that target mHSP 90.

Some embodiments disclose biological materials comprising nucleic acid molecules encoding a polypeptide that targets mHSP90, including recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineering bacteria, or transgenic cell lines.

Some embodiments disclose a DNA fragment comprising an amino acid sequence encoding polypeptide P7.

Some embodiments disclose the use of a polypeptide that targets mHSP90, comprising:

(1) For preparing medicines for treating diseases related to abnormal mHSP90 expression; drugs are generally used for preventing or treating diseases associated with abnormal expression of mHSP 90;

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

In some embodiments, the mHSP 90-targeting polypeptide is used to prepare a prophylactic or therapeutic agent for a disease associated with abnormal expression of a mHSP, the agent comprising the mHSP-targeting polypeptide, an agent for killing cancer cells coupled to the mHSP-targeting polypeptide, and optionally a drug carrier; the preparation for killing cancer cells is chemical medicine, biological medicine, nanometer medicine, radioactive medicine, photothermal therapy or photodynamic therapy medicine for killing cancer cells.

In some preferred embodiments, the agent for killing cancer cells is an alkylating agent, an antimetabolite, an antineoplastic agent, an antineoplastic antibiotic, an antineoplastic neovascular agent, a metal complex, or a tumor-targeting marker.

In some embodiments, the polypeptide that targets msp 90 is used to prepare a molecular imaging agent for a disease associated with abnormal msp expression for use 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.

Some embodiments disclose the use of a polypeptide that targets mHSP90, wherein the disease associated with abnormal mHSP90 expression is a tumor, including 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.

Some embodiments disclose a molecular probe targeting mHSP90, formed by linking a signal 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, and an ultrasonic microbubble to a polypeptide targeting mHSP 90.

Some embodiments disclose molecular probes targeting mHSP90 wherein the polypeptide targeting mHSP90 is linked to the signal unit by a linking molecule, the linking means comprising mixing with a multimer, or non-covalent linking, or direct linking of the signal unit to the polypeptide by other chemical reactions; wherein the linking molecule comprises thioether, maleimidocaproyl, disulfide bond, hydrazone bond, valine-citrulline dipeptide bond, amide, 6-t-butoxycarbonylhydrazinonicotinic acid, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide or N-hydroxysuccinimide; for example, 6-t-butoxycarbonyl hydrazinonicotinic acid reacts with the Pro carboxyl group at the C-terminus of the polypeptide; reacting 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide or N-hydroxysuccinimide with the carboxyl group at the C-terminus of the polypeptide; or long chain diacid is connected with the N end of Pro; valine-citrulline dipeptide bond and disulfide bond are introduced at two ends of the P7 polypeptide; the polymer is any one or a combination of at least two of polyethylene glycol (PEG), polyvinyl alcohol (PVA), cyclodextrin, polyamide-amine dendritic Polymer (PAMAM), polylactic acid (PLA) and polylactic acid-ethanolamine (PLGA).

Some embodiments disclose a molecular probe targeting mHSP90, wherein the polypeptide targeting mHSP90 is linked to the signal unit by a chelator comprising DTPA, DOTA, NOTA or TETA; typically the amino group at the N-terminus of the polypeptide is coupled by reaction with the carboxyl group of the chelator.

Some embodiments disclose molecular probes targeting mHSP90 for use in the preparation of molecular imaging agents for diseases associated with abnormal mHSP90 expression.

In some embodiments, conjugates of a polypeptide targeting mHSP90 with chelators DTPA and DOTA may chelate paramagnetic Gd for MRI imaging; conjugates of polypeptides with chelators DTPA, DOTA, NOTA or TETA may chelate ⁶⁸ Ga or ⁶⁴ Cu was PET imaged.

Some embodiments disclose molecular probes targeting mHSP90 for use in the preparation of reagents for diagnosing diseases associated with abnormal mHSP90 expression, as medicaments or imaging agents for immunotherapy, prevention or diagnosis of cancer.

In some embodiments, a targeting molecular probe that targets tumor cell membrane-bound mHSP90 comprises a signaling unit, a targeting affinity unit, and a linker for linking the signaling unit and the targeting affinity unit, wherein the signaling unit is a moiety that is detectable by an imaging device and the targeting affinity unit is a polypeptide moiety that specifically binds to mHSP 90.

In some embodiments, the mHSP 90-targeted molecular probes can be used to perform fluorescence imaging, positron emission tomography, single photon emission tomography, magnetic resonance imaging, photoacoustic imaging, ultrasound imaging, or other fused imaging techniques for living imaging of a patient.

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

Some embodiments disclose the use of a molecular probe that targets mHSP90, where the disease associated with abnormal mHSP90 expression is a tumor, including 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.

Further exemplary details are described below in connection with the embodiments.

Example 1

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

The preparation and fluorescence detection of the FITC-P7 polypeptide fluorescent molecular probe specifically comprise the following steps:

weighing 100mg of wang-Cys resin, and sequentially circulating according to the sequence of the P7 polypeptide according to a solid-phase polypeptide synthesis strategy;

deprotection and cleaning are carried out after the coupling reaction is finished;

adding epsilon-aminocaproic acid to perform room temperature reaction for 2 hours, and after deprotection, mixing Fluorescein Isothiocyanate (FITC) with 50mg of resin in a solution of pyridine, N dimethylformamide and dichloromethane in a volume ratio of 1:2:4, and performing light-shielding room temperature reaction overnight, wherein the mole ratio of FITC to resin is 1:1;

after the reaction is finished, cleaning the methanol with isopropanol for dehydration;

removing side chain protecting groups from the lysate in the resin, and vacuum pumping to obtain crude polypeptide FITC fluorescent conjugate;

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

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

Example 2

Preparation of ICG-P7, cy-5 and Cy-7 polypeptide imaging preparation and functional verification thereof

This example 2 exemplifies the preparation of ICG-P7 polypeptide fluorescent conjugates, and illustrates the preparation and functional verification of P7 polypeptide near infrared in vivo imaging and imaging agents. The method specifically comprises the following steps:

1mg of the synthesized P7 polypeptide prepared in example 1 is weighed and dissolved in 1 XPBS, 0.5mg of indocyanine green-maleimide (ICG-MAL) is weighed and dissolved in 500 mu L of deionized water, the two are mixed, the pH is regulated to about 7.4, the shaking reaction is carried out at room temperature for 24 hours, and mass spectrum detection and HPLC purification are carried out after freeze drying;

the obtained product is dialyzed and freeze-dried to obtain an ICG-polypeptide imaging preparation which is used for fluorescence imaging of mice.

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

1mg of ICG-P7 polypeptide is weighed and dissolved in 1mL of 1 XPBS, 150 mu L of ICG-polypeptide is injected into tail vein for half an hour, and then an IVIS Spectrum small animal living body optical three-dimensional imaging system is used for signal acquisition;

and taking the main organ after 24h dissection for in vitro fluorescence distribution imaging.

As a result, the result is shown in FIG. 3, and ICG was successfully labeled on the P7 polypeptide by MALDI-TOF-MS identification.

The polypeptide imaging preparation prepared in the example 2 is injected into a tumor-bearing mouse through a tail vein, as shown in fig. 4, ICG-P7 is obviously enriched at a tumor part at a 5-hour time point, ICG is only slightly enriched at the tumor part, the fluorescence of the ICG-P7 group mouse at the tumor part is gradually weakened at 24-hour and 48-hour time points, and the tumor part of the ICG group mouse does not show fluorescence, so that ICG-P7 has good targeting to in-vivo tumors and can be excreted from the body through metabolism with time.

Example 3

Fluorescence probe absorption spectrum and emission spectrum detection

The specific method for detecting the absorption wavelength comprises the following steps:

the Cy5/Cy5-P7 stock was diluted to a concentration of 5. Mu.M using PBS;

cy5 and Cy5-P7 were added to a transparent bottom 96-well plate at 200. Mu.l/well, three wells were parallel and placed in the dark;

turning on a fluorescence microplate reader, selecting absorption spectrum scanning, setting parameters to be in a detection range of 400-700 nm, and setting a detection step length to be 1nm;

click number, absorbance values for each well were read.

The emission spectrum detection method comprises the following steps:

the Cy5-P7 stock was diluted to a concentration of 5. Mu.M with PBS;

cy5 and Cy5-P7 were added to a black 96-well plate at 200 μl/well, three wells were parallel and placed in the dark;

turning on a fluorescence microplate reader, selecting emission spectrum scanning, setting parameters to be 600nm of excitation wavelength, 620nm-700nm of detection range and 1nm of detection step length;

click number, read fluorescence value for each well.

The results showed that there was no significant change in the absorption and emission spectra of the fluorescent probe after coupling P7, indicating that the fluorescent properties of the fluorescent probe were not changed after coupling P7, as shown in FIG. 5.

Example 4

Flow cytometry detection of uptake of molecular probes by tumor cells

The specific method comprises the following steps:

the cell concentration was adjusted to 2X 105/ml;

adding 1 ml/hole of cell suspension into a six-hole plate, supplementing the cell suspension to 2ml by using a culture solution, and placing the cell suspension into an incubator for culture overnight;

the following day the culture broth was discarded and 2ml serum-free DMEM high-sugar broth containing 10nM Cy5/Cy5-P7 was added at 37℃for 1h;

discarding the culture solution containing the fluorescent probe, washing for 3 times by using PBS, adding pancreatin without EDTA for digestion for 4min, blowing the cells and transferring the cells into a 1.5ml centrifuge tube, centrifuging for 5min at 600g, and gently sucking the supernatant;

adding 500 μl PBS, blowing and mixing, centrifuging at 600g for 5min, and discarding supernatant;

the cells were resuspended by adding 500 μl PBS and detected on-line by flow cytometry.

The results showed that the average fluorescence intensity in the final cells was measured using flow cytometry using the same molar concentration of fluorochrome for each of the PANC-1 cells, and that the average fluorescence intensity in the cells incubated with Cy5 was similar to that in the control group, whereas the average fluorescence intensity in the cells incubated with Cy5-P7 was significantly enhanced, as shown in FIG. 6.

Example 5

Blocking study of uptake of molecular probes of tumor cells

The specific method comprises the following steps:

the original culture solution is discarded, 2ml of fresh culture solution is added, 20 mu l of Hsp90 monoclonal antibody AC88 is added into an AC88 antibody blocking group, the final concentration is 10 mu g/ml, and the culture solution is incubated for 30min at 4 ℃;

the original culture solution is reserved, and Cy5/Cy5-P7 liquid medicine is added into each hole according to groups, wherein the final concentration is 10nM, and the temperature is 37 ℃ for 1h;

blocking the uptake process of Cy5-P7 by using an Hsp90 monoclonal antibody, and detecting by a flow cytometry method and a fluorescence staining method respectively;

the results showed a significant decrease in the average fluorescence intensity in the cells after blocking, as shown in fig. 6.

Example 6

Immunofluorescence staining method for detecting uptake of Cy5-P7 by PANC-1 cells

The specific method comprises the following steps:

preparing PANC-1 cells into 2X 105/ml cell suspension;

placing a cover glass subjected to high-pressure sterilization in a six-hole plate, manufacturing a cell climbing sheet, adding 1 ml/hole of cell suspension, supplementing 2ml with culture solution, and placing in an incubator for overnight culture;

discarding the original culture solution, washing for 2 times by PBS, and adding 2ml of serum-free DMEM high-sugar culture medium;

20 μl of Hsp90 monoclonal antibody AC88 was added to the AC88 antibody blocking group at a final concentration of 10 μg/ml, and incubated at 4deg.C for 30min; the original culture solution is reserved, and Cy5/Cy5-P7 liquid medicine is added into each hole according to groups, wherein the final concentration is 10 mu M, and the temperature is 37 ℃ for 1h;

discarding culture solution, washing with PBS for 3min,2 times, adding 4% paraformaldehyde, and incubating at room temperature for 15min;

absorbing and discarding paraformaldehyde, washing with PBS for 3min,2 times, adding DAPI dye liquor, and keeping away from light at room temperature for 30min;

sucking DAPI dye liquor, and cleaning with ultrapure water for 3min for 2 times;

the excess liquid was wiped off, capped with an anti-fluorescence quencher, and immediately observed with a confocal microscope or stored at 4 ℃.

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

Example 7

Synthesis method of DOTA-P7 for nuclear magnetic resonance detection

The 2-CTC resin is used as starting resin, fmoc solid phase method is adopted to gradually couple amino acids Fmoc-Pro-OH, fmoc-Leu-OH, fmoc-Pro-OH, fmoc-Thr-OH, fmoc-Leu-OH, fmoc-Pro-OH and Fmoc-Leu-O, TUBU/HOBt/DIEA is selected as an amino acid condensing agent, after the amino acid coupling is finished, fmoc is removed, and TristBu-DOTA (1, 4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetra-tert-butyl tetraacetate)/TBTU/HOBt/DIEA is used for end sealing. The reaction is controlled by K detection/C detection after each step of reaction, if the condensation reaction of certain amino acid is incomplete, the condensation is repeated once, the re-addition equivalent of the amino acid is 1.0eq until the required target peptide fragment is obtained, and the peptide resin is washed and dried after coupling.

And (3) using TFA lysate to crack peptide resin, removing side chain protecting groups, controlling the temperature to be 20-30 ℃, settling by isopropyl ether, drying to obtain DOTA-P7 crude product, purifying in one step, and converting salt in two steps to obtain DOTA-P7 finished product. The structural formula is shown in figure 7.

The polypeptide of the targeting mHSP90 disclosed by the embodiment of the invention can be used for carrying out high expression on cell membrane-bound HSP90, and can be used for preparing a detection reagent for diseases related to abnormal mHSP90 expression and a molecular imaging agent for preparing the diseases related to abnormal mHSP90 expression; the molecular probe of the targeted mHSP90 can be used for preparing a molecular imaging agent of a disease related to abnormal mHSP90 expression, has the characteristics of low cost, small molecular weight, good biocompatibility, strong penetrability, no immunogenicity, high blood clearance rate, simple preparation and the like, and has very strong superiority in tumor targeted drug delivery, cancer diagnosis and the like of pancreatic cancer, lung cancer, nerve tumor, breast cancer, gastric cancer, colorectal cancer, liver cancer, osteosarcoma, cholangiocarcinoma, thyroid cancer, angiosarcoma, gastric cancer, ovarian cancer, cervical cancer or prostate cancer, and has good application prospect.

The technical solutions disclosed in the embodiments of the present invention and the technical details disclosed in the embodiments of the present invention are only exemplary to illustrate the inventive concept of the present invention, and do not constitute a limitation on the technical solutions of the embodiments of the present invention, and all conventional changes, substitutions or combinations of the technical details disclosed in the embodiments of the present invention have the same inventive concept as the present invention, and are within the 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.

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