CN108570094A - AE polypeptides and its purposes in preparing cancer target diagnosis and treatment delivery system - Google Patents

Abstract

AE polypeptide and its use in preparing tumor-targeted drug delivery system for diagnosis and treatment. The invention belongs to the field of pharmacy, and relates to D-configuration polypeptide ^D AE and L-configuration polypeptide which are highly stable and can target epidermal growth factor receptor and epidermal growth factor receptor mutant III high-expression cells, and are used in the preparation of tumor diagnosis and target The application of diagnostic and therapeutic drug complexes, modified polymer carrier materials and their constructed liposomes, polymer micelles, polymer discs, nanoparticles and other drug delivery systems. The test results show that: ^D AE's EGFR protein and EGFRvIII protein binding activity is more stable in serum; the modified drug is specifically taken up by positive cells and tumor tissues expressing EGFR or EGFRvIII, and has good tumor targeting and imaging functions; The constructed nano drug delivery system can effectively deliver the drug to the target tissue, and the active targeting effect in vivo is better, which has a good application prospect in tumor diagnosis and targeted therapy.

Description

AE polypeptide and its use in the preparation of tumor-targeted diagnosis and treatment drug delivery system

technical field

The invention belongs to the field of pharmacy, and relates to AE polypeptide and its application in the preparation of drug delivery system for tumor targeting diagnosis and treatment, in particular to highly stable and targeted epidermal growth factor receptor and epidermal growth factor receptor mutant III high-expressing cells D-configuration polypeptides, drug complexes of D-configuration polypeptides and L-configuration polypeptides, and modified nano drug delivery systems, especially involving D-configuration polypeptides ^D AE (D-configuration amino acid sequence ^D F ^D A ^D L ^D G ^D E ^D A), and L configuration polypeptide ^L AE (L configuration amino acid sequence FALGEA) and D configuration polypeptide ^D AE in the preparation of diagnostic and therapeutic drug complexes for tumor diagnosis and targeted therapy, modified polymer carrier materials and Applications in drug delivery systems such as liposomes, polymer micelles, polymer discs, and nanoparticles constructed by it.

Background technique

Data show that tumor is a disease that seriously threatens human life and health, and its mortality rate ranks first among all diseases. As the main means of tumor drug treatment, traditional chemotherapy has defects such as poor selectivity to tumor tissue, high toxicity, narrow therapeutic window, and easy multidrug resistance. Therefore, in order to overcome the limitations of chemotherapy, active targeting has become An important strategy to improve tumor tissue targeting efficiency. The active targeting strategy mainly targets highly expressed receptors or transporters in tumor tissues, and uses corresponding ligands that have the ability to recognize and bind specific receptors or transporters to deliver drugs or nano drug delivery systems into tumor tissues or cells . Commonly used corresponding ligands include monoclonal antibodies, polypeptides, nucleic acid aptamers, small molecule compounds, etc. Studies have shown that ligand-modified drugs or nano-drug delivery systems can achieve specificity through cell surface receptors or transporters and ligands. Recognize, bind, internalize, and deliver drugs to tumor tissues and cells, thereby achieving active targeting of tumors.

Epidermal growth factor receptor (EGFR) is a specific receptor for epidermal growth factor, and the EGFR signal transduction pathway plays an important role in tumor cell proliferation, damage repair, invasion and angiogenesis. In most tumors (such as breast cancer, glioma, prostate cancer, non-small cell lung cancer, etc.), EGFR is overexpressed and accompanied by mutation and rearrangement. Studies have reported that 40-50% of glioma cells have abnormally increased EGFR accompanied by a variety of gene mutations. The most common mutation is epidermal growth factor receptor mutant III (EGFRvIII), and the extracellular part 2-7 of EGFR is overexpressed. The EGFRvIII formed by the lack of ligand-dependent autophosphorylation can generate continuous transduction signals and promote the proliferation, invasion and migration of glioma cells; ^L AE (L configuration amino acid sequence FALGEA) is a mixture The hexapeptides with high binding activity to EGFR and EGFRvIII screened out from the position scanning library can target tumor neovascularization, mimic blood vessels and tumor cells with high expression of EGFR and EGFRvIII. Reporting to medical treatment.

Contents of the invention

The purpose of the present invention is to provide the application of AE polypeptide in tumor targeting diagnosis and treatment and further optimize the stability of the existing polypeptide to achieve better tumor targeting effect in vivo. It is specifically related to the preparation of highly stable D configuration polypeptide ^D AE (D configuration amino acid sequence ^D F ^D A ^D L ^D G ^D E ^D A), and modified with ^L AE (L configuration amino acid sequence FALGEA) and ^D AE Drug molecules and polymer carrier materials to construct AE drug complexes and AE-modified nano drug delivery systems to achieve targeted diagnosis and treatment of tumors.

Specifically, the present invention utilizes solid-phase polypeptide synthesis technology to design and prepare a D-configuration polypeptide ^D AE, which has high stability to serum and is compatible with epidermal growth factor receptor (EGFR) and EGFR mutant III (EGFRvIII) with high affinity.

In the present invention, after ^L AE and the designed ^D AE are connected to cysteine, the fluorescent substance (such as Fluorescein, near-infrared dye Cy5.5, IR820, DiR, magnetic Resonant imaging agent Gd-DTPA, radiographic agent ^99m Tc-DTPA, etc.) react to form complexes;

In the present invention, ^LAE and designed ^D AE modified drugs include forming pH-sensitive hydrazone bonds through the reaction of maleimide hexylhydrazine derivatives (related to drugs containing ketone or aldehyde groups such as doxorubicin and epirubicin) ), or through the reaction of 3-(2-pyridyldimercapto)propionic acid derivatives to form disulfide bonds (involving paclitaxel, docetaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, vincristine Hydroxyl or amino group-containing drugs), or through the reaction of dopamine and boronic acid groups in drugs to form pH-sensitive borate lipids (involving bortezomib and other boronic acid-containing drugs), or directly forming amide bonds by solid-phase synthesis (involving p53 Activating peptides, antimicrobial peptides, polypeptide toxins and other polypeptide drugs) peptide-drug complexes;

In the present invention, after ^{the LAE} and the designed ^D AE are linked to cysteine, they are modified in polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) containing maleimide functional groups, polyethylene glycol Glycol-polylactic acid (PEG-PLA), polyethylene glycol-lactic acid glycolic acid copolymer (PEG-PLGA), polyethylene glycol-polycaprolactone (PEG-PCL) and other polymer carrier materials, can be used for Construct ^LAE and ^D AE modified liposomes, polymer micelles, polymer discs, nanoparticles and other drug delivery systems.

The nano drug delivery system modified by ^LAE and ^DAE designed by the present invention contains paclitaxel, docetaxel, doxorubicin, epirubicin, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, Vincristine, bortezomib, carfilzomib, parthenolide, p53 activating peptide, melittin, scorpion venom and other anti-tumor drugs; fluorescent substances coumarin 6, FAM, near-infrared Dyes Cy5.5, IR820, DiR, DiD, magnetic resonance imaging agent Gd-DTPA and other imaging substances.

^{The DAE} designed in the present invention can mediate the drug or nano drug delivery system to target cells and tissues with high expression of EGFR and EGFRvIII, so as to be used for targeted diagnosis and treatment of tumors.

The present invention provides the material basis for the preparation and property investigation of ^DAE and the above-mentioned ^LAE and ^DAE modified drug complexes and nano drug delivery systems for tumor diagnosis and treatment; the test results of the present invention show that: ^{both LAE} and ^DAE can be mediated active targeting in vivo; compared with ^LAE , ^DAE has better stability in serum, so its active targeting effect mediated in vivo is better. In the embodiments of the present invention, the following technical solutions are disclosed:

1. Synthesis of AE, AE-Cys and their fluorescent markers (AE-Fluorescein, AE-Cy7)

^{The LAE} , ^LAE -Cys, ^DAE , and ^DAE -Cys were prepared by a solid-phase peptide synthesis method. Synthesis of ^LAE -Fluorescein, ^DAE -Fluorescein, ^LAE -Cy7, ^DAE -Cy7 by Michael addition reaction of maleimide group and sulfhydryl group; structure characterized by HPLC and MS;

2. Synthesis of AE-DTPA-Gd and AE-DTPA- ^99m Tc

Synthesize ^DAE -DTPA or ^LAE -DTPA through the Michael addition reaction of maleimide group and sulfhydryl group, and chelate Gd or ^99m Tc to obtain AE-DTPA-Gd or AE-DTPA- ^99m Tc;

3. Evaluation of AE stability and receptor affinity

The properties of ^DAE were investigated from the aspects of serum stability and the uptake ability of cells with high expression of EGFR and EGFRvIII. ^DAE and ^LAE were incubated with mouse serum at 37°C, and the concentration of peptides was detected at different time points. Comparison of stability, comparing the in vitro targeting of ^D AE-Fluorescein and ^LAE -Fluorescein to cells with high expression of EGFR and EGFRvIII (such as: umbilical vein endothelial cells HUVEC) and model tumor cells (such as: glioma cells U87) To compare the uptake ability of ^D AE-Fluorescein and ^L AE-Fluorescein in the in vitro 3D tumor sphere model;

4. Preparation of AE drug complexes

^{The LAE} and ^D AE linked to cysteine react with the maleimide hexylhydrazine derivative of the drug to form a polypeptide-drug complex containing a pH-sensitive hydrazone bond. The drugs involved include doxorubicin, epi-A Drugs containing ketones or aldehyde groups such as mycin;

^{The LAE} and ^D AE linked to cysteine react with the 3-(2-pyridinedimercapto)propionic acid derivative of the drug to form a polypeptide-drug complex containing a disulfide bond, wherein the drugs involved include paclitaxel, poly Hydroxyl or amino-containing drugs such as paclitaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, and vincristine;

^LAE and ^D AE modify dopamine and then react with the boronic acid group of the drug to form a polypeptide-drug complex containing pH-sensitive borate lipids. The drugs involved include bortezomib and other boronic acid group-containing drugs;

^L AE and ^D AE are directly condensed with polypeptide drugs through solid-phase synthesis to make fusion polypeptides, and the drugs involved include polypeptide drugs such as p53 activating peptides, antibacterial peptides, and polypeptide toxins;

5. Construction and characterization of AE liposome drug delivery system

Firstly, polymer materials ^D AE-PEG-DSPE and ^L AE-PEG-DSPE modified by ^D AE ^and L AE were synthesized. ^DAE -PEG-DSPE and ^LAE -PEG-DSPE are obtained by connecting free sulfhydryl groups on ^DAE -Cys and ^LAE -Cys with Mal-PEG-DSPE;

Then prepare ^DAE and ^LAE modified liposomes ( ^DAE -Liposome and ^LA7R -Liposome) respectively, with a certain ratio of HSPC/Chol/mPEG ₂₀₀₀ -DSPE/AE-PEG-DSPE as membrane material and drug (FAM , DiR or doxorubicin), using membrane-forming hydration method to prepare liposomes (AE-Liposome/FAM, AE-Liposome/DiR or AE-Liposme/DOX), and reducing the size of liposomes by extrusion Particle size, construct liposomes with an average particle size of about 100nm. Laser scattering particle size analyzer to characterize micellar particle size and particle size distribution;

6. Construction and characterization of AE micellar drug delivery system

Firstly, ^L AE and ^D AE modified polymer materials ^L AE-PEG-PLA and ^D AE-PEG-PLA were synthesized. The reaction of amines realizes the synthesis of materials, and is characterized by ¹ H-NMR;

^LAE and ^DAE micelles ( ^LAE -Micelle, ^DAE -Micelle) were then prepared. A certain amount of AE-PEG-PLA, mPEG-PLA and drugs (coumarin-6, DiR or paclitaxel) were prepared by film-forming micelles (AE-Micelle/C6, AE-Micelle/DiR or AE-Micelle/PTX ), the laser scattering particle size meter characterizes the micellar particle size and particle size distribution;

7. In vivo and in vitro tumor targeting evaluation of AE-Micelle

Investigate the uptake of ^LAE -Micelle/C6, ^D AE-Micelle/C6 and mPEG-Micelle/C6 by the in vitro tumorsphere model of U87 cells;

^L AE-Micelle/DiR, ^D AE-Micelle/DiR, and mPEG-Micelle/DiR were injected into the tail vein of nude mice bearing U87 subcutaneous xenograft tumor model, and the tumor distribution of different groups at each time point was compared;

8. In vivo anti-tumor effect evaluation of AE-Micelle/PTX

^L AE-Micelle/PTX, ^D AE-Micelle/PTX, mPEG-Micelle/PTX, clinical preparation Taxol, and normal saline were respectively injected into the tail vein of nude mice bearing U87 subcutaneously transplanted tumor model, and the tumor volume, tumor weight and tumor The number of tissue cell apoptosis, new blood vessels and mimic blood vessels were used as indexes to evaluate the in vivo anti-tumor effect of different paclitaxel-loaded micelles.

The invention provides a drug complex modified by ^L AE polypeptide and a modified nano-drug delivery system, which can realize targeted diagnosis and treatment of tumors; at the same time, the L-configuration polypeptide is easily degraded in blood, which may lead to a decrease in tumor targeting ability The problem is to provide a highly stable D-configuration polypeptide targeting molecule ^D AE (D-configuration amino acid sequence ^D F ^D A ^D L ^D G ^D E ^D A) with high binding activity to EGFR and EGFRvIII, and construct its modified Drug complexes and modified nano drug delivery systems have achieved better tumor targeting and diagnosis and treatment effects in vivo.

Description of drawings

Figure 1, HPLC and ESI-MS spectra of ^D AE

Chromatographic method: chromatographic column (YMC, C18): 150×4.6mm; mobile phase A: water (containing 0.1% trifluoroacetic acid), mobile phase B: acetonitrile (containing 0.1% trifluoroacetic acid); elution procedure: 0- 30min 5%B-65%B; flow rate: 0.7mL/min; column temperature: 40°C; detection: UV 214nm, retention time: 13.95min, ESI-MS: 606, consistent with the theoretical molecular weight.

Figure 2. HPLC and ESI-MS spectra of ^D AE-Cys

Chromatographic method is the same as above, retention time: 14.33min. ESI-MS: 709.2, consistent with the theoretical molecular weight.

Figure 3. HPLC and ESI-MS spectra of ^L AE

Chromatographic method is the same as above, retention time: 13.95min. ESI-MS: 606, consistent with the theoretical molecular weight.

Figure 4. HPLC and ESI-MS spectra of ^L AE-Cys

Chromatographic method is the same as above, retention time: 14.33min. ESI-MS: 709.2, consistent with the theoretical molecular weight.

Figure 5. HPLC and ESI-MS spectra of ^D AE-Fluorescein

Chromatographic method is the same as above, retention time: 18.63min. ESI-MS: 1137.18, consistent with the theoretical molecular weight.

Figure 6. HPLC and ESI-MS spectra of ^L AE-Fluorescein

Chromatographic method is the same as above, retention time: 18.63min. ESI-MS: 1137.18, consistent with the theoretical molecular weight.

Figure 7, HPLC and ESI-MS spectra of ^D AE-Cy7

Chromatographic method is the same as above, retention time: 31.21min. ESI-MS: 1416.66, consistent with the theoretical molecular weight.

Figure 8. HPLC and ESI-MS spectra of ^L AE-Cy7

Chromatographic method is the same as above, retention time: 31.21min. ESI-MS: 1416.66, consistent with the theoretical molecular weight.

Figure 9, ¹ H-NMR spectra of ^D AE-PEG-DSPE and ^L AE-PEG-DSPE

The NMR spectrum of Mal-PEG-DSPE shows a maleimide peak at 6.7ppm, while this peak disappears in the NMR spectrum of ^L AE-PEG-DSPE and ^D AE-PEG-DSPE, showing that the maleimide in Mal-PEG-DSPE The imide groups are attached to AE.

Figure 10, ¹ H-NMR spectra of ^D AE-PEG-PLA and ^L AE-PEG-PLA

The NMR spectrum of Mal-PEG-PLA shows a maleimide peak at 6.7ppm, while this peak disappears in the NMR spectrum of ^L AE-PEG-PLA and ^D AE-PEG-PLA, showing that the maleimide in Mal-PEG-PLA The imide groups are attached to AE.

Figure 11. Particle size of liposomes loaded with doxorubicin

The figure is the particle diameter of each liposome loaded with doxorubicin, as can be seen from the figure, there is no significant difference in the liposome size and shape of each prescription.

Serum stability of Fig. 12, ^D AE and ^LAE

The vertical axis of the figure is the residual percentage of the complete polypeptide. It can be seen that the stability of ^DAE in 50% mouse serum is significantly higher than that of ^LAE . After incubation for 2 hours, ^LAE is completely degraded and ^DAE is almost not degraded.

Figure 13. Uptake of Fluorescein-labeled polypeptide by glioma cell U87

Among them, the left and right pictures are laser confocal photos and flow cytometry fluorescence detection results of Fluorescein-labeled ^DAE and ^LAE after 4 hours of interaction with U87 cells. It can be seen that the uptake of ^DAE and ^LAE by U87 cells is significantly higher than that of U87 cells. free fluorescein, but there was no significant difference in the uptake of the two peptides.

Figure 14. Uptake of Fluorescein-labeled polypeptide by HUVEC in umbilical vein endothelial cells

Among them, the left picture and the right picture are respectively the laser confocal photos and flow cytometry fluorescence detection results of Fluorescein-labeled ^DAE and ^LAE interacting with HUVEC cells for 4 hours. It can be seen that the uptake of ^DAE and ^LAE by HUVEC cells is significantly higher than that of free fluorescein, but there is no significant difference in the uptake of the two polypeptides.

Figure 15. Uptake of Fluorescein-labeled polypeptide by U87 in vitro tumorsphere model

The picture shows the fluorescent tomographic images of ^DAE and ^LAE labeled with ^Fluorescein and the U87 in vitro tumorsphere model for 4 hours ^. There was no significant difference in the uptake of these peptides.

Figure 16. Distribution of Cy7-labeled polypeptide in nude mice with subcarcinoma xenografts

Among them, Figure A is the image distribution result of the isolated tumor after the tail vein injection of Cy7-labeled polypeptide in nude mice bearing U87 subcutaneously transplanted tumor for 2 hours; Figure B is the fluorescence semi-quantitative result of the isolated tumor; Figure C is the image of the isolated tumor and organs. Fluorescence distribution images; Figure D is the fluorescence semi-quantitative results of isolated tumors and organs, the results show that the accumulation of Cy7-labeled ^DAE and ^LAE in tumors is significantly higher than that of free Cy7 (***p<0.001), The tumor targeting effect of ^D AE is better than that of ^LAE .

Figure 17. Uptake of C6-loaded micelles by U87 in vitro tumorsphere model

The picture shows the fluorescence tomographic images of ^D AE-Micelle/C6, ^L AE-Micelle/C6, mPEG ^- Micelle/C6 and U87 in vitro tumor sphere ^model cells after 30 minutes of interaction. The uptake was significantly higher than that of unmodified micelles.

Figure 18. In vivo distribution of subcutaneous tumor nude mice loaded with near-infrared fluorescent dye micelles

Among them, Figure A is the in vivo fluorescence distribution image of DiR micelles loaded with near-infrared fluorescent dye injected into the tail vein at various time points; Figure B is the fluorescence distribution image of isolated tumors and organs after administration of the preparation for 24 hours; Figure C is the fluorescence distribution image of Figure B The semi-quantitative statistical results show that, compared with mPEG-Micelle, ^D AE-Micelle and ^L AE-Micelle can better target the tumor site, and the targeting effect of ^D AE-Micelle is better than that of ^L AE-Micelle.

Figure 19. Particle size of paclitaxel-loaded micelles

The picture shows the particle size of each paclitaxel-loaded micelles. It can be seen from the figure that there is no significant difference in the size and shape of the micelles in each prescription.

Figure 20. Activity curve of paclitaxel-loaded micelles against U87 cells and HUVEC cells in vitro

Among them, Figures A and B are the activity curves of mPEG-Micelle/PTX, ^DAE -Micelle/PTX, ^LAE -Micelle/PTX and Taxol against U87 cells and HUVEC cells, respectively. Panel A shows that after U87 cells were administered for 4 hours and cultured for 72 hours, their IC ₅₀ were 0.21, 0.02, 0.05 and 0.31 nM, respectively. All three micelles could inhibit the growth of U87 cells in vitro, and the in vitro activity of ^D AE-Micelle/PTX was 2.5 times that of ^L AE-Micelle/PTX. Panel B shows that HUVEC cells were cultured for 72 hours after administration for 4 hours, and their IC ₅₀ were 0.70, 0.06, 0.21 and 0.85 nM, respectively. All three micelles could inhibit the growth of HUVEC cells in vitro, and the in vitro activity of ^DAE -Micelle/PTX was 3.5 times that of ^LAE -Micelle/PTX.

Figure 21. Inhibition of paclitaxel-loaded micelles on an in vitro model of neovascularization

Among them, Figure A is mPEG-Micelle/PTX, ^L AE-Micelle/PTX, ^D AE-Micelle/PTX and Inhibition photos of neovascularization model in vitro, Figure B is the quantitative result of neovascularization rate, compared with mPEG-Micelle/PTX, ^D AE-Micelle/PTX and ^LAE -Micelle/PTX inhibit the formation of neovascularization more significantly, and The inhibitory effect of ^D AE-Micelle was better than that ^{of L} AE-Micelle (*p<0.05, **p<0.01).

Figure 22. Inhibition of paclitaxel-loaded micelles on the mimic blood vessel model in vitro

Among them, Figure A is mPEG-Micelle/PTX, ^L AE-Micelle/PTX, ^D AE-Micelle/PTX and Inhibition photos of mimic vessel models in vitro, Figure B shows the quantitative results of the formation rate of mimic vessel structures in each group, compared with mPEG-Micelle/PTX, ^D AE-Micelle/PTX and ^LAE -Micelle/PTX inhibited the formation of mimic vessels More significantly, and the inhibitory effect of ^D AE-Micelle is better than that ^{of L} AE-Micelle (*p<0.05, ***p<0.001).

Figure 23. Inhibition effect of paclitaxel-loaded micelles on subcutaneous transplanted tumors

Graph A is the curve of the tumor volume of nude mice in each group changing with time. Compared with the normal saline group, each administration group has an inhibitory effect on tumor growth. Compared with mPEG-Micelle/PTX, both ^DAE -Micelle/PTX and ^LAE -Micelle/PTX could significantly inhibit tumor growth, and the antitumor efficacy of ^DAE -Micelle/PTX was significantly better than that of ^L AE-Micelle/PTX( n=8, ***p<0.001), Figure B shows that the nude mice were killed and the tumor tissues were taken out and weighed and statistically analyzed. The results showed that the tumor size of each group was: ^D AE-Micelle /PTX< ^L AE-Micelle /PTX<mPEG-Micelle/PTX, ^D AE-Micelle/PTX has the most significant tumor inhibitory effect (n=8, ***p<0.001).

Figure 24. TUNEL staining results

The picture shows mPEG-Micelle/PTX, ^L AE-Micelle/PTX, ^D AE-Micelle/PTX and TUNEL staining photos of subcutaneous tumors (bar=100μm), in which the nuclei are brownish yellow or brown as positive apoptotic cells, compared with mPEG-Micelle/PTX, ^D AE-Micelle/PTX and ^LAE -Micelle/PTX promote tumor Tissue apoptosis was more significant, and the pro-apoptotic effect of ^D AE-Micelle was better than that of ^L AE-Micelle.

Figure 25, CD31/PAS double staining results

The picture shows mPEG-Micelle/PTX, ^L AE-Micelle/PTX, ^D AE-Micelle/PTX and CD31/PAS double-stained photos (bar=100μm), the nuclei are brownish yellow or brown for new blood vessels, compared with mPEG-Micelle/PTX, ^D AE-Micelle/PTX and ^LAE -Micelle/PTX inhibited the growth of new blood vessels The formation was more significant, and the anti-angiogenesis effect of ^D AE-Micelle was better than that of ^L AE-Micelle.

Detailed ways

The following examples will help to further understand the present invention, but the present invention is not limited to the scope of the following description.

Example 1

Synthesis and characterization of AE, AE-Fluorescein, AE-Cy7, AE-DTPA-Gd, AE-DTPA- ^99mTc , AE-drug, AE-PEG-PLA

1) Synthesis and characterization of ^L AE and ^D AE, ^L AE-Cys and ^D AE-Cys

DAE (sequence: ^{D F D} A ^D L ^D G ^D E ^D A) and ^DAE -Cys (sequence: ^D F ^D A ^D L ^D G ^D E ^D A ^D C) and ^LAE (sequence is FALGEA) and ^LAE -Cys (sequence is FALGEAC) composed of L-configuration amino acids.

Specific method: using the Boc solid-phase peptide synthesis method, insert amino acids in sequence on the PAM resin, and react with HBTU/DIEA as the condensing agent and TFA as the deprotecting agent. After the reaction was completed, the resin was cut with hydrogen fluoride containing P-cresol, and the reaction was stirred in an ice bath for 1 h. After the reaction, the hydrogen fluoride was removed under reduced pressure, and the precipitate was precipitated with glacial ether and washed 3 times. The precipitate was redissolved with 20% acetonitrile, and the filtrate was collected. After rotary evaporation, the crude polypeptide solution was obtained, and the crude polypeptide was separated and purified by the acetonitrile/water (containing 0.1% TFA) system. HPLC and ESI-MS characterize the purity and molecular weight (Mw) of ^DAE , ^DAE -Cys, ^LAE and ^LAE -Cys, HPLC collection of illustrative plates, mass spectrogram as shown in Figure 1, Figure 2, Figure 3 and Figure 4;

2) Synthesis and characterization of AE-Fluorescein and AE-Cy7

Dissolve ^{the D} AE-Cys or ^L AE-Cys prepared above in 0.1M PBS solution (pH7.2), dissolve Fluorescein-5-maleimide in DMF, mix the two and react with magnetic stirring, monitor by HPLC, and wait for the reaction After the preparation is completed, the liquid phase is purified, separated and purified with acetonitrile/water (containing 0.1% TFA), and freeze-dried to obtain pure ^D AE-Fluorescein or ^L AE-Fluorescein. The HPLC spectrum and mass spectrum are shown in Figures 5 and 6;

The preparation method of AE-Cy7 is the same as above, and the HPLC spectrum and mass spectrum are shown in Figures 7 and 8;

3) Preparation of AE-DTPA-Gd and AE-DTPA- ^99m Tc

Maleimide-DTPA was dissolved in DMF, mixed with the PBS solution of ^D AE-Cys or ^L AE-Cys as above, stirred and reacted, purified by preparative liquid phase, and freeze-dried to obtain pure ^D AE-DTPA or ^L AE-DTPA, chelating Gd or ^99m Tc is AE-DTPA-Gd or AE-DTPA- ^99m Tc;

4) Preparation of AE-drug complexes, including,

Prepare AE-doxorubicin complex as an example of AE-linked ketone or ^aldehyde -containing drug ^; ), adding 10-fold molar amount of tris(2-carboxyethyl)phosphine (TCEP), and stirring at 4° C. for 20 min. Then add 4-fold molar amount of doxorubicin 6-maleimide hexylhydrazine derivative, and react at room temperature for 1 h in the dark. The reaction solution was purified by preparative liquid phase and freeze-dried to obtain ^DAE or ^LAE -doxorubicin complex.

Take the AE-paclitaxel complex as an example of AE linking hydroxyl or amino-containing drugs with disulfide bonds; 200mg of paclitaxel was dissolved in 10mL of chloroform, cooled to 0-5°C, and 39.99mg of DCC and 60.4mg of 3-(2-pyridine were added successively Dimercapto)propionic acid, after the addition was completed, it was raised to room temperature and reacted overnight, the reaction solution was filtered, and purified by column chromatography (CHCl ₃ /MeOH=50:1-15:1, V/V elution) to obtain paclitaxel 3-( 2-pyridine dimercapto) propionic acid derivatives, paclitaxel 3-(2-pyridine dimercapto) propionic acid derivatives were dissolved in 5mL DMF, 1.5 times the molar amount of AE-Cys was dissolved in PBS/DMF, and the pH of the solution was kept 4-5, add paclitaxel 3-(2-pyridyldithiol) propionic acid derivative dropwise to the thiol polypeptide solution, react at room temperature for 6 hours, and obtain the AE-paclitaxel complex through preparative liquid phase purification and lyophilization;

Taking the AE-bortezomib complex as an example of AE linking boronic acid group-containing drugs, insert amino acids on the resin in sequence according to the synthesis of AE. After all the amino acid residues of the polypeptide are attached, trifluoroacetic acid removes the Boc at the nitrogen end Protect. Add a DMF solution containing 3 times the molar amount of succinic anhydride and DIEA, and react at room temperature for 30 minutes. After washing the resin, add 5 times molar amount of trimethylchlorosilane to protect dopamine, and use HBTU/DIEA as condensing agent to react at room temperature for 1 h. The resin was cleaved with HF and purified by preparative HPLC to obtain AE-dopamine derivatives. In a buffer solution with pH 7.4, AE-dopamine derivatives were mixed with bortezomib at a molar ratio of 1:1 to obtain AE-bortezomib Complex;

Taking the AE-PMI fusion polypeptide as an example of an AE-linked polypeptide drug, it is directly prepared by solid-phase peptide synthesis. The specific method is: after determining the sequence of the AE-PMI polypeptide, insert amino acids in sequence in the same way as the preparation of AE, and cut with HF And after purification, AE-PMI fusion polypeptide is obtained;

5) Synthesis and characterization of AE-PEG-DSPE

Dissolve ^D AE-Cys or ^L AE-Cys in 0.1M PBS solution (pH7.2), take Mal-PEG-PLA and dissolve it in DMF. After mixing the two, magnetically stir the reaction, monitor by HPLC, and wait until Mal-PEG-PLA Stop the reaction after the DSPE reaction is complete, and remove excess ^DAE -Cys, ^LAE -Cys and DMF by dialysis (molecular weight cut-off 3.5kDa), freeze-dry to obtain ^DAE -PEG-DSPE or ^LAE -PEG-DSPE, and characterize its structure by NMR (as shown in Figure 9);

6) Synthesis and characterization of AE-PEG-PLA

Dissolve ^D AE-Cys or ^L AE-Cys in 0.1M PBS solution (pH7.2), take Mal-PEG-PLA and dissolve it in DMF. After mixing the two, magnetically stir the reaction, monitor by HPLC, and wait until Mal-PEG-PLA Stop the reaction after the PLA reaction is complete, and remove excess ^DAE -Cys, ^LAE -Cys and DMF by dialysis (molecular weight cut-off 3.5kDa), freeze-dry to obtain ^DAE -PEG-PLA or ^LAE -PEG-PLA, and characterize its structure by NMR (as shown in Figure 10).

The preparation of embodiment 2 AE liposome/Dox

The prescription composition of PEG-liposome membrane material is HSPC/Chol/mPEG ₂₀₀₀ -DSPE (52:43:5, mol/mol), and the prescription of peptide-modified PEG liposome membrane material is HSPC/Chol/mPEG ₂₀₀₀ -DSPE/ Polypeptide-PEG-DSPE (52:43:3:2, mol/mol), weigh the above membrane material and dissolve it in chloroform, remove the organic solvent by rotary evaporation under reduced pressure to obtain a uniform lipid film, dry it in vacuum for 24 hours, and use ammonium sulfate gradient Each liposome loaded with doxorubicin (DOX) was prepared by the method, and the particle size distribution was determined by the dynamic light scattering method (as shown in FIG. 11 ).

The serum stability investigation of embodiment 3 AE

Make ^D AE and ^L AE into 1mg/mL aqueous solution, take 0.1mL and add it to 0.9mL 50% mouse serum, incubate at 37°C, at 0, 5 and 15min, 0.5, 1, 2, 4, 6 and 8h respectively Take out 100 μL, add 20 μL trichloroacetic acid (TCA) to precipitate the protein in the serum, let it stand at 4°C for 20 minutes, centrifuge at 12000 rpm for 10 minutes, take 20 μL of the supernatant for HPLC analysis, the serum stability results (as shown in Figure 12) show that , ^DAE has better serum stability than ^LAE .

Example 4 In vitro cell targeting verification of AE

1) In vitro targeting of AE to glioma cell U87

Take the monolayer cultured glioma cells (U87 cells) in the logarithmic growth phase, digest the monolayer cultured cells with 0.25% trypsin, and prepare a single cell suspension with DMEM culture medium containing 10% fetal bovine serum. 1× ¹⁰⁵ cells per well were inoculated in a 12-well culture plate, the volume of each well was 1 mL, the culture plate was moved into a carbon dioxide incubator, and cultured for 24 hours at 37 ° C, 5% CO ₂ and saturated humidity conditions, with 10% fetal Prepare FAM, ^D AE-Fluorescein and ^L AE-Fluorescein solutions with a concentration of 5 μM in the DMEM culture solution of bovine serum, suck out the culture solution in the culture plate, add the above solutions respectively, incubate at 37°C for 4 hours, discard the supernatant, and use Wash three times with PBS solution, fix the cells with 4% paraformaldehyde fixative solution, stain the nuclei with DAPI, and observe with laser confocal. , the result is shown in the right figure of Figure 13;

2) In vitro targeting of AE to human umbilical vein endothelial cells HUVEC

Take monolayer cultured human umbilical vein endothelial cells (HUVEC cells) in the logarithmic growth phase, and perform the same experiment. The photos of cell internalization are shown in the left figure of Figure 14, and the results of flow cytometry analysis are shown in the right figure of Figure 14;

3) Targeting of AE to U87 in vitro tumorsphere model

Add 150 μL of Agrose gel solution to each well of a 48-well culture plate, irradiate it under UV light for 30 minutes and wait for it to solidify, inoculate 400 μL of U87 cell suspension in each well with a cell density of 2×10 ³ cells/well, and place in a carbon dioxide incubator. Tumorspheres were formed after being cultured at 37°C, 5% CO ₂ and saturated humidity for 7 days. FITC, ^DAE -Fluorescein and ^LAE -Fluorescein solutions with a concentration of 5 μM were prepared with DMEM medium containing 10% fetal bovine serum. Aspirate the culture solution in the culture plate, add the above solution respectively, incubate at 37°C for 4 hours, discard the supernatant, wash with PBS solution three times, fix with paraformaldehyde, and observe with a fluorescence microscope. The photos are shown in Figure 15.

Example 5 In vivo tumor targeting verification of AE

Firstly, a subcutaneous tumor animal model was constructed, U87 cells in the logarithmic growth phase were digested with trypsin, the cell concentration was adjusted to 3×10 ⁷ cells/mL, and 100 μL was inoculated into the subcutaneous area near the axilla on the right dorsal side of nude mice. After inoculation, they were raised in SPF grade, regularly observe the tumor size, when the tumor size is ^200mm3 , select tumor-bearing nude mice with no necrosis and regular tumor shape, and conduct experiments in groups, 200 μL of Cy7, ^DAE -Cy7 and ^LAE -Cy7 with the same fluorescence intensity The solution was injected into the tumor-bearing nude mouse animal model through the tail vein, and the nude mice were sacrificed 2 hours later, and the tumor was removed. The distribution of fluorescence in the tumor-bearing nude mice was detected with an in vivo imager and the fluorescence was semi-quantitatively calculated. The results are shown in Figure 16.

Example 6 In vitro targeting verification of AE-Micelle

1) Preparation of AE-Micelle/C6 micelles

The prescription of AE-Micelle is 9mg mPEG-PLA, 1mg AE-PEG-PLA, weigh the above micellar material and 5ug coumarin-6 and dissolve in 2ml acetonitrile, 37℃ water bath, vacuum (~0.085MPa) rotary evaporation Remove the organic solvent to form a uniform film, dry it under vacuum at room temperature overnight, add 2ml of normal saline for hydration, remove unencapsulated C6 by CL-4B column chromatography, and obtain AE-Micelle/C6;

2) Targeting of AE-Micelle to U87 in vitro tumorsphere model

Prepare mPEG-Micelle/C6, ^D AE-Micelle/C6, and ^L AE-Micelle/C6 solutions with a C6 concentration of 5 μM in DMEM culture medium containing 10% fetal bovine serum. Aspirate, add the above solutions respectively, incubate at 37°C for 30 minutes, discard the supernatant, wash with PBS solution three times, fix with paraformaldehyde, and observe with a fluorescence microscope. The results are shown in Figure 17.

Example 7 In vivo targeting verification of AE-Micelle

1) Preparation of AE-Micelle/DiR

AE-Micelle/DiR was prepared in the same way as AE-Micelle/C6;

2) In vivo targeting verification of AE-Micelle

Inject 100 μL of mPEG-Micelle/DiR, ^D AE-Micelle/DiR, and ^L AE-Micelle/DiR solutions into the tail vein, anesthetize the mice at 4, 6, 8, 12, and 24 hours after injection, and record them with an in vivo imager. Fluorescence distribution in tumor-bearing nude mice was performed and fluorescence semi-quantitative calculation was performed, and the results are shown in FIG. 18 .

Example 8 In vitro pharmacodynamics test of AE-Micelle/PTX

1) Preparation and characterization of AE-Micelle/PTX

AE-Micelle/PTX was prepared in the same manner as AE-Micelle/C6. Laser scattering particle size meter characterizes micellar particle size and particle size distribution (as shown in Figure 19);

2) In vitro efficacy test of AE-Micelle/PTX

Inoculate U87 cells or HUVEC cells in 96-well plates at 4.0×10 ³ cells/well. After 24 hours, suck out the culture medium and add 200 μL of mPEG-Micelle/PTX, ^D AE-Micelle/PTX, ^L AE- For Micelle/PTX and Taxol, after administration for 4 hours and incubation for 72 hours, add MTT solution to continue incubation for 4 hours, discard the culture solution, add 150 μL DMSO, shake until the purple particles are dissolved, measure the absorbance value at 590nm with a microplate reader, and use the MTT method Determination of cell viability, calculation of the median lethal dose, the results are as shown in Figure 20;

3) Inhibition test of AE-Micelle/PTX on neovascularization

Add 50 μL Matrigel to each well of a 24-well culture plate, spread it in a 24-well plate, incubate in a 37°C incubator for 30 minutes until it solidifies, digest HUVEC cells with 0.25% trypsin, and use micelles containing 1 μM paclitaxel or free paclitaxel The DMEM culture medium of the solution was made into a single cell suspension, and 1×10 ⁵ cells per well were inoculated in a 24-well culture plate, and cultured at 37°C, 5% CO ₂ and saturated humidity for 12 hours to observe the formation of blood vessel-like structures ( As shown in Figure 21A) and calculate the formation rate of blood vessel-like structures (as shown in Figure 21B);

4) Inhibition test of AE-Micelle/PTX on mimic vessel formation

Add 50 μL Matrigel to each well of a 24-well culture plate, spread it in a 24-well plate, incubate in a 37°C incubator for 30 minutes until it solidifies, digest U87 cells with 0.25% trypsin, and use micelles containing 1 μM paclitaxel or free paclitaxel The DMEM culture medium of liquid was prepared into a single cell suspension, and 1× ¹⁰⁵ cells per well were inoculated in a 24-well culture plate, and cultured at 37°C, 5% CO ₂ and saturated humidity for 12 hours to observe the formation of blood vessel-like structures ( 22A) and calculate the formation rate of the mimic vessel structure (as shown in FIG. 22B).

Example 9 In vivo efficacy test of AE-Micelle/PTX

1) Inhibition test of AE-Micelle/PTX on subcutaneous xenograft tumor

The U87 subcutaneous tumor animal model was constructed, and the tumor size was regularly observed. ^When the tumor size was 100mm, the experiment was performed in groups, and mPEG-Micelle/PTX, ^D AE-Micelle/PTX, ^L AE-Micelle/PTX, commercially available Taxol and normal saline were each 100μl, and the total dosage of PTX in the administration group was 25mg/kg, which was divided into five times, and the interval between each administration was two days. Diameter (b), calculate the tumor volume of nude mice in each group according to the formula, draw the change curve of tumor volume with time, and calculate the statistical difference of each group,

Tumor volume calculation formula: Vtumor _volume =0.5(a×b ² )

After 15 days of administration (21 days after inoculation), all nude mice were killed by neck dislocation, and the subcutaneous tumors were taken and weighed, and the statistical differences among the groups were calculated (as shown in Figure 23);

2) AE-Micelle/PTX pro-apoptosis assay

After the five doses of administration, the tumor-bearing nude mice were sacrificed, the tumor tissues were taken out, fixed, embedded in paraffin and sectioned. Terminal deoxynucleotidyl transferase (TDT)-mediated dUTP nick end labeling (Terminal deoxynucleotidylTransferase-mediated dUTP nick end labeling, TUNEL) was used to detect the degree of apoptosis of tumor cells, and the cell nucleus was judged to be apoptosis when it was brown or brown. Apoptotic cells were continuously observed under an optical microscope to count positive cells in three high-power fields, and the results are shown in Figure 24;

3) AE-Micelle/PTX inhibition test on tumor blood vessels

After five doses of administration, the tumor-bearing nude mice were sacrificed, the subcutaneous tumors were taken out, fixed in paraffin, and sectioned for CD31 immunohistochemical staining and PAS double staining. Angiogenesis was observed under an optical microscope. The results are shown in Figure 25.

Claims (17)

1.AE polypeptides, which is characterized in that it includes D configuration polypeptides^DAE, wherein D amino acids sequences are^DF^DA^DL^DG^DE^DA, and L-configuration polypeptide^LAE, wherein L-configuration amino acid sequence is FALGEA.

2. AE polypeptides described in claim 1, which is characterized in that D configurations polypeptide therein^DAE is passed preparing cancer target diagnosis and treatment Purposes in medicine system.

3. purposes as described in claim 2, which is characterized in that the D configuration polypeptides^DAE while targeting epidermal growth factor Receptor and epidermal growth factor receptor mutations body III, for drug or the targeted delivery of nanoscale medicine delivery system.

4. purposes as described in claim 2, which is characterized in that the D configuration polypeptides^DAE mediates EGF-R ELISA With epidermal growth factor receptor mutations body III, the cancer target delivering of drug molecule or nano medicament carrying system is realized.

5. a kind of^DAE-X compounds, which is characterized in that use D configurations polypeptide described in claim 1^DAfter AE sulfhydrylations with contain The image substance reaction of maleimide base group obtains.

6. a kind of^LAE-X compounds, which is characterized in that using described in claim 1^LAfter AE sulfhydrylations and contain maleimide The image substance reaction of amine groups obtains；Described^LAE-X compounds while targeting epidermal growth factor receptor and epidermal growth Factor acceptor mutant III.

7. by described in claim 5 or 6^DAE-X compounds or^LAE-X compounds, which is characterized in that X is selected in the compound Autofluorescence substance Fluorescein, nir dye Cy7, IR820, DiR, nuclear magnetic resonance image agent Gd-DTPA or irradiation image Agent^99mTc-DTPA, the image for high expression EGF-R ELISA or epidermal growth factor receptor mutations body III tumours are examined Disconnected and tracer.

8. AE polypeptides as described in claim 1, which is characterized in that D configurations polypeptide therein^DHydrazone bonds of the AE by pH sensitivities, pH Sensitive boric acid fat key, disulfide bond and medicine connects, or fused polypeptide directly is made with polypeptide drugs condensation, obtains^DAE- Y compounds；

L-configuration polypeptide therein^LAE is connected by the hydrazone bond of pH sensitivities, the boric acid fat key of pH sensitivities, disulfide bond and medicine, Or fused polypeptide directly is made with polypeptide drugs condensation, it obtains^LAE-Y compounds.

9. AE polypeptides as described in claim 9, which is characterized in that described^DAE-Y compounds or^LY is anti-in AE-Y compounds Anti-neoplastic drug doxorubicin, Epi-ADM, taxol, Docetaxel, camptothecine, hydroxycamptothecin, 9-nitrocamptothecin, Changchun New alkali, bortezomib, parithenolide, p53 activating peptides, melittin or scorpion venom peptide.

10. AE polypeptides as described in claim 1, which is characterized in that D configurations polypeptide therein^DAfter AE sulfhydrylations and contain Malaysia Polyethylene glycol-Z the compounds of imide group connect, and obtain^DAE- polyethylene glycol-Z compounds；

It is therein^LIt connect, obtains with the polyethylene glycol-Z compounds of maleimation after AE sulfhydrylations^LAE- polyethylene glycol-Z is multiple Close object.

11. AE polypeptides as described in claim 10, which is characterized in that described^DAE- polyethylene glycol-Z compounds or and^LAE- Z is selected from phosphatide, polylactic acid (PLA), lactic-co-glycolic acid (PLGA) or polycaprolactone in polyethylene glycol-Z compounds (PCL)。

12. by the AE polypeptides described in claim 11, which is characterized in that wherein obtained^DAE- polyethylene glycol-phosphorus fat complexes Or^LAE- polyethylene glycol-phosphorus fat complexes are being used to prepare liposome delivery systems, polymer micelle delivery system or polymer Purposes in disk delivery system.

13. by the AE polypeptides described in claim 11, which is characterized in that wherein obtained^DAE- polyethylene glycol-polylactic acids are compound Object,^DAE- polyethylene glycol-lactic-co-glycolic acid compound,^DAE- polyethylene glycol-polycaprolactone compound,^LThe poly- second of AE- Glycol-polylactic acid composition,^LAE- polyethylene glycol-lactic-co-glycolic acid compound or^LAE- polyethylene glycol-polycaprolactone Purposes of the compound in being used to prepare polymer micelle delivery system or nanoparticle delivery system.

14. by the AE polypeptides described in claim 12 or 13, which is characterized in that liposome delivery systems made from described, polymerization Object micella delivery system, polymer disc delivery system or nanoparticle delivery system are for containing diagnostic medicine.

15. by the AE polypeptides described in claim 14, which is characterized in that the diagnostic medicine is selected from fluorescent material cumarin 6, FAM, nir dye Cy7, IR820, DiR, DiD or nuclear magnetic resonance image agent Gd-DTPA, for high expression epidermal growth factor The diagnostic imaging and tracer of receptor or epidermal growth factor receptor mutations body III tumours.

16. by the AE polypeptides described in claim 12 or 13, which is characterized in that liposome delivery systems made from described, polymerization Object micella delivery system, polymer disc delivery system or nanoparticle delivery system contain antitumor drug.

17. by the AE polypeptides described in claim 16, which is characterized in that the antitumor drug is selected from adriamycin, table Ah mould Element, taxol, Docetaxel, camptothecine, hydroxycamptothecin, 9-nitrocamptothecin, vincristine, bortezomib, Ka Feizuo Rice, parithenolide, p53 activating peptides, melittin or scorpion venom peptide, for high expression EGF-R ELISA or epidermal growth factor The targeted therapy of sub- acceptor mutant III tumours.