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

AE polypeptides and its purposes in preparing cancer target diagnosis and treatment delivery system Download PDF

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CN108570094A
CN108570094A CN201710144712.4A CN201710144712A CN108570094A CN 108570094 A CN108570094 A CN 108570094A CN 201710144712 A CN201710144712 A CN 201710144712A CN 108570094 A CN108570094 A CN 108570094A
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polypeptides
micelle
delivery system
compounds
polypeptide
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陆伟跃
毛佳妮
冉丹妮
谢操
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Fudan University
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Fudan University
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    • C—CHEMISTRY; METALLURGY
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Abstract

AE polypeptides and its purposes in preparing cancer target diagnosis and treatment delivery system.The invention belongs to pharmaceutical field, be related to it is highly stable and can targeting epidermal growth factor receptor and epidermal growth factor receptor mutations body III high expressing cells D configuration polypeptidesDAE and L-configuration polypeptide, and the application in preparing the delivery systems such as the diagnosing and treating medicinal composition of diagnosing tumor and targeted therapy, the polymer carrier of modification and its constructed liposome, polymer micelle, polymer disc, nanoparticle.It is shown through test result：DThe EGFR albumen and EGFRvIII protein binding activities of AE is more stable in serum；The drug modified is expressed positive cell and the tumor tissue specificity intake of EGFR or EGFRvIII, has good cancer target and imaging function；The nanoscale medicine delivery system of structure can effectively deliver the medicament to target tissue, and internal active targeting effect is more excellent, has good application prospect in diagnosing tumor and targeted therapy.

Description

AE polypeptides and its purposes in preparing cancer target diagnosis and treatment delivery system
Technical field
The invention belongs to pharmaceutical fields, are related to AE polypeptides and its purposes in preparing cancer target diagnosis and treatment delivery system, tool Body is related to highly stable and can targeting epidermal growth factor receptor and epidermal growth factor receptor mutations body III high expressing cells D configurations polypeptide and D configurations polypeptide and L-configuration polypeptide medicinal composition and modification nanoscale medicine delivery system more particularly to D Configuration polypeptideDAE (D amino acids sequencesDFDADLDGDED) and L-configuration polypeptide ALAE (L-configuration amino acid sequence FALGEA) and D configuration polypeptidesDAE is preparing the diagnosing and treating medicinal composition of diagnosing tumor and targeted therapy, the macromolecule carrier material of modification Application in the delivery systems such as material and its constructed liposome, polymer micelle, polymer disc, nanoparticle.
Background technology
Data shows that tumour is to seriously threaten the disease of human life and health, and the death rate is in all mortalities It is the first.Main means of traditional chemotherapy as tumor pharmacother exist to tumor tissues poor selectivity, toxicity is big, treats Window is narrow, is also easy to produce the defects of multidrug resistance, and therefore, to overcome the limitation of chemotherapy, in recent years, active targeting, which becomes, improves tumour The Critical policies of tissue targeting efficiency.Active targeting strategy is mainly for the receptor or transporter of high expression in tumor tissues, profit With with specific receptor or transporter have identification, binding ability corresponding ligand, drug or nanoscale medicine delivery system are delivered to In tumor tissues or cell.Common corresponding ligand includes monoclonal antibody, polypeptide, aptamer, micromolecular compound etc., is ground Study carefully display, it is ligand modified after drug or nanoscale medicine delivery system can pass through the specificity of cell surface receptor or transporter and ligand Identification, combination, internalization deliver the medicament to tumor tissues and intracellular, to realize the active targeting to tumour.
EGF-R ELISA (EGFR) is the specific receptor of epidermal growth factor, and EGFR signal transduction pathways are swollen Proliferation, injury repair, invasion and new vessels formation of oncocyte etc. play an important role.(such as breast in most of tumours Gland cancer, glioma, prostate cancer, non-small cell lung cancer etc.), EGFR, which exists, to be overexpressed and with mutation, rearrangement.Research report, The glioma cell of 40-50% increases there are EGFR and is mutated with several genes extremely, and most common mutant form is epidermis The EGFRvIII that the missing of growth factor receptor mutations body III (EGFRvIII), EGFR extracellular portion 2-7 exons is formed, can By the autophosphorylation of non-ligand-dependent, lasting transduction signal is generated, promotes the proliferation of glioma cell, invade and move It moves；LAE (L-configuration amino acid sequence FALGEA) be by mix object location scan base filter out with EGFR and EGFRvIII There is height to combine active hexapeptide, tumor neogenetic blood vessels, mimicry blood vessel and the tumour of energy targeting EGFR and EGFRvIII high expression Cell, but so far, there is not yet carrying out the report in terms of cancer target diagnosis and treatment for mediate drug.
Invention content
The application in cancer target diagnosis and treatment that the object of the present invention is to provide AE polypeptides simultaneously advanced optimizes existing polypeptide Stability reaches better in-vivo tumour Targeting Effect.More particularly to D configuration polypeptide of the preparation with high stabilityDAE (D structures Type amino acid sequenceDFDADLDGDEDA), and withLAE (L-configuration amino acid sequence FALGEA) andDAE modified medicaments molecule and high score Subcarrier material, the nanoscale medicine delivery system that structure AE medicinal compositions, AE are modified, to realize targeting diagnosis and treatment of the drug to tumour.
Specifically, the present invention utilizes Solid phase peptide synthssis technology, designs and be prepared for D configuration polypeptidesDAE, the polypeptide is to blood There is clearly high stability and EGF-R ELISA (EGFR) and epidermal growth factor receptor mutations body III (EGFRvIII) With high-affinity.
In the present invention,LAE with it is designedDAfter AE connection cysteines, sulfydryl in its molecule and maleimide work(are utilized Fluorescent material (such as Fluorescein, nir dye Cy5.5, IR820, DiR, nuclear magnetic resonance image agent Gd-DTPA, radiation can be changed Imaging agents99mTc-DTPA etc.) reaction form compound；
In the present invention,LAE and designedDAE modified medicaments, including react to be formed by the own hydrazine derivate of maleimide PH sensitivities hydrazone bond (being related to the drug containing ketone or aldehyde radical such as adriamycin, Epi-ADM) passes through 3- (2- pyridines dimercapto) propionic acid Derivatives reaction forms disulfide bond and (is related to taxol, Docetaxel, camptothecine, hydroxycamptothecin, 9-nitrocamptothecin, length The drug of the hydroxyls such as spring new alkali or amino) or by dopamine react to form pH sensitivity boric acid fat with boric acid base group in drug (drug for being related to the boracics acid groups such as bortezomib) or directly formed by synthesis in solid state amido bond (be related to p53 activating peptides, The polypeptide drugs such as antibacterial peptide, polypeptide toxin) polypeptide-medicinal composition；
In the present invention,LAE and designedDAfter AE connection cysteines, the poly- second in the base of amine functions containing maleimide is modified Glycol-distearoylphosphatidylethanolamine (PEG-DSPE), polyethylene glycol-polylactic acid (PEG-PLA), polyethylene glycol-lactic acid On the polymer carriers such as co-glycolic acid (PEG-PLGA), polyethylene glycol-polycaprolactone (PEG-PCL), it can be used for StructureLAE andDThe delivery systems such as liposome, polymer micelle, polymer disc, the nanoparticle of AE modifications.
Designed by the present inventionLAE andDAE modification nanoscale medicine delivery system contain taxol, Docetaxel, adriamycin, Epi-ADM, camptothecine, hydroxycamptothecin, 9-nitrocamptothecin, vincristine, boron are in azoles rice, Carfilzomib, feverfew The antitumor drugs such as ester, p53 activating peptides, melittin, scorpion venom peptide；Also fluorescent material coumarin 6, FAM, nir dye can be contained The images substances such as Cy5.5, IR820, DiR, DiD, nuclear magnetic resonance image agent Gd-DTPA.
Designed by the present inventionDAE can mediate drug or nanoscale medicine delivery system targeting EGFR and EGFRvIII high expression Cell and its tissue, the targeting diagnosis for tumour and treatment.
The present invention providesDPrepared by AE and property is investigated and above-mentionedLAE andDThe medicinal composition and nanometer that AE is modified Delivery system is used for the material base of tumour diagnosis and treatment；The test result of the present invention shows：LAE andDThe internal active that AE can be mediated Targeting；WithLAE is compared,DAE stability in serum is more preferable, thus its internal active targeting effect mediated is more excellent.The present invention's In embodiment, following technical proposals are disclosed：
The synthesis of 1.AE, AE-Cys and its fluorescent marker (AE-Fluorescein, AE-Cy7)
It is prepared using solid-phase peptide synthesisLAE、LAE-Cys、DAE、DAE-Cys.Pass through maleimide base group and mercapto The Michael addition reactions of base synthesizeLAE-Fluorescein、DAE-Fluorescein、LAE-Cy7、DAE-Cy7；HPLC、MS Characterize structure；
2.AE-DTPA-Gd and AE-DTPA-99mThe synthesis of Tc
It is synthesized with the Michael addition reactions of sulfydryl by maleimide base groupDAE-DTPA orLAE-DTPA chelates Gd Or99mTc obtains AE-DTPA-Gd or AE-DTPA-99mTc；
3.AE stability and receptor affinity evaluation
It is carried out in terms of serum stability, with the high cellular uptake ability two for expressing EGFR and EGFRvIIIDAE properties It investigates, it willDAE andLAE is incubated with mice serum at 37 DEG C respectively, and the concentration for detecting polypeptide in different time points is stablized The comparison of property, comparesDAE-Fluorescein、LThe cell that AE-Fluorescein expresses EGFR and EGFRvIII high is (such as： Huve cell HUVEC) and Model Tumor Cells are (such as：Brain glioblastoma cell U87) targeting, the outer 3D of comparing bulk Tumour spherical model pairDAE-Fluorescein、LThe intake ability of AE-Fluorescein；
The preparation of 4.AE medicinal compositions
After connecting cysteineLAE andDAE is reacted with the own hydrazine derivate of the maleimide of drug, is formed sensitive containing pH Polypeptide-medicinal composition of hydrazone bond, wherein involved drug includes the drug containing ketone or aldehyde radical such as adriamycin, Epi-ADM；
After connecting cysteineLAE andDAE is reacted with 3- (2- pyridines dimercapto) propanoic derivatives of drug, and formation contains Polypeptide-medicinal composition of disulfide bond, wherein involved drug includes taxol, Docetaxel, camptothecine, hydroxy-camptothecin The drug of the hydroxyls such as alkali, 9-nitrocamptothecin, vincristine or amino；
LAE andDAE is reacted by modification upper dopamine with the boric acid base group of drug, and the boric acid fat of sensitivity containing pH is formed Polypeptide-medicinal composition, wherein involved drug includes the drug of the boracics acid groups such as bortezomib；
LAE andDFused polypeptide directly is made with polypeptide drugs condensation by synthesis in solid state in AE, wherein involved drug includes The polypeptide drugs such as p53 activating peptides, antibacterial peptide, polypeptide toxin；
The structure and characterization of 5.AE liposome delivery systems
It synthesizes firstDAE、LThe high molecular material of AE modificationsDAE-PEG-DSPE andLAE-PEG-DSPE.Pass through connectionDAE- Cys andLFree sulfhydryl groups on AE-Cys are obtained by the reaction with Mal-PEG-DSPEDAE-PEG-DSPE andLAE-PEG-DSPE；
Then it prepares respectivelyDAE andLAE modification liposome (DAE-Liposome andLA7R-Liposome), centainly to compare The HSPC/Chol/mPEG of example2000- DSPE/AE-PEG-DSPE is membrane material and drug (FAM, DiR or adriamycin), is used Film forming aquation method prepares liposome (AE-Liposome/FAM, AE-Liposome/DiR or AE-Liposme/DOX), with extruding The method for crossing film reduces liposomal particle size, liposome of the structure average grain diameter in 100nm or so.Laser diffraction particle size instrument levies glue Beam grain size and particle diameter distribution；
The structure and characterization of 6.AE micella delivery systems
It synthesizes firstLAE andDThe high molecular material of AE modificationsLAE-PEG-PLA andDAE-PEG-PLA, by connecting half Guang The synthesis for reacting realization material of free sulfhydryl groups and maleimide contained by Mal-PEG-PLA after propylhomoserin on polypeptide,1H-NMR Characterization；
Then it preparesLAE andDAE micellas (LAE-Micelle、DAE-Micelle).A certain amount of AE-PEG-PLA, MPEG-PLA and drug (Coumarin-6, DiR or taxol) prepare micella (AE-Micelle/C6, AE- using membrane formation process Micelle/DiR or AE-Micelle/PTX), laser diffraction particle size instrument levies micella grain size and particle diameter distribution；
The inside and outside tumor-targeting evaluation of 7.AE-Micelle
Investigate U87 cells in vitro tumour spherical models pairLAE-Micelle/C6、DAE-Micelle/C6 and mPEG- The intake situation of Micelle/C6；
It is injected respectively by lotus U87 subcutaneous transplantation knurl model nude mice tail veinsLAE-Micelle/DiR、DAE-Micelle/ DiR and mPEG-Micelle/DiR, more different groups are distributed in the tumour at each time point；
The internal antitumous effect of 8.AE-Micelle/PTX is evaluated
It is injected respectively by lotus U87 subcutaneous transplantation knurl model nude mice tail veinsLAE-Micelle/PTX、DAE-Micelle/ PTX, mPEG-Micelle/PTX, clinical preparation taxol, physiological saline, with knurl product, knurl weight and tumor tissue cell's apoptosis, New vessels and the internal antitumous effect that mimicry blood vessel number is that metrics evaluation difference carries taxol micella.
The present invention providesLThe nanoscale medicine delivery system of medicinal composition and modification peptide modified AE, can realize tumour Targeting diagnosis and treatment；It is degradable in blood for L-configuration polypeptide simultaneously, asking for cancer target ability reduction may be caused Topic provides highly stable and has height to combine active D configurations polypeptide targeted molecular with EGFR and EGFRvIIIDAE (D configurations Amino acid sequenceDFDADLDGDEDA), and build its modification medicinal composition and modification nanoscale medicine delivery system, obtain more preferable In-vivo tumour targeting and treatment effect.
Description of the drawings
Fig. 1,DHPLC the and ESI-MS collection of illustrative plates of AE
Chromatographic process：Chromatographic column (YMC, C18)：150×4.6mm；Mobile phase A：Water (contains 0.1% trifluoroacetic acid), flowing Phase B：Acetonitrile (contains 0.1% trifluoroacetic acid)；Elution program：0-30min 5%B-65%B；Flow velocity：0.7mL/min；Column temperature：40 ℃；Detection：UV 214nm, retention time：13.95min ESI-MS：606, it is consistent with theoretical molecular weight.
Fig. 2,DHPLC the and ESI-MS collection of illustrative plates of AE-Cys
Chromatographic process is same as above, retention time：14.33min.ESI-MS：709.2, it is consistent with theoretical molecular weight.
Fig. 3,LHPLC the and ESI-MS collection of illustrative plates of AE
Chromatographic process is same as above, retention time：13.95min.ESI-MS：606, it is consistent with theoretical molecular weight.
Fig. 4,LHPLC the and ESI-MS collection of illustrative plates of AE-Cys
Chromatographic process is same as above, retention time：14.33min.ESI-MS：709.2, it is consistent with theoretical molecular weight.
Fig. 5,DHPLC the and ESI-MS collection of illustrative plates of AE-Fluorescein
Chromatographic process is same as above, retention time：18.63min.ESI-MS：1137.18 being consistent with theoretical molecular weight.
Fig. 6,LHPLC the and ESI-MS collection of illustrative plates of AE-Fluorescein
Chromatographic process is same as above, retention time：18.63min.ESI-MS：1137.18 being consistent with theoretical molecular weight.
Fig. 7,DHPLC the and ESI-MS collection of illustrative plates of AE-Cy7
Chromatographic process is same as above, retention time：31.21min.ESI-MS：1416.66 being consistent with theoretical molecular weight.
Fig. 8,LHPLC the and ESI-MS collection of illustrative plates of AE-Cy7
Chromatographic process is same as above, retention time：31.21min.ESI-MS：1416.66 being consistent with theoretical molecular weight.
Fig. 9,DAE-PEG-DSPE andLAE-PEG-DSPE's1H-NMR collection of illustrative plates
The nuclear magnetic spectrum of Mal-PEG-DSPE shows maleimide peak in 6.7ppm, andLAE-PEG-DSPE、DAE- The peak disappears in PEG-DSPE nuclear magnetic spectrums, shows that the maleimide base group in Mal-PEG-DSPE has connected AE.
Figure 10,DAE-PEG-PLA andLAE-PEG-PLA's1H-NMR collection of illustrative plates
The nuclear magnetic spectrum of Mal-PEG-PLA shows maleimide peak in 6.7ppm, andLAE-PEG-PLA、DAE-PEG- The peak disappears in PLA nuclear magnetic spectrums, shows that the maleimide base group in Mal-PEG-PLA has connected AE.
Figure 11, the grain size for carrying Evacet
Figure is each grain size for carrying Evacet, and as seen from the figure, each prescription liposome size and form are without significance difference It is different.
Figure 12,DAE andLThe serum stability of AE
Figure ordinate is the residual percentage of complete polypeptide, it is seen thatDStability of the AE in 50% mice serum is significantly high InLAE is incubated 2h,LAE is degradable,DAE is almost non-degradable.
The intake of Figure 13, brain glioblastoma cell U87 to Fluorescein labeling polypeptides
Wherein, left figure and right figure are respectively what Fluorescein was markedDAE andLLaser after AE and U87 cytosiies 4h The burnt photo of copolymerization and fluidic cell fluoroscopic examination result, it is seen that U87 cells pairDAE andLThe intake of AE is apparently higher than free fluorescence Element, but to the no notable difference of the intake of two kinds of polypeptides.
The intake of Figure 14, huve cell HUVEC to Fluorescein labeling polypeptides
Wherein, left figure and right figure are respectively what Fluorescein was markedDAE andLSwashing after AE and HUVEC cytosiies 4h The burnt photo of light copolymerization and fluidic cell fluoroscopic examination result.It can be seen that HUVEC cells pairDAE andLThe intake of AE is apparently higher than free Fluorescein, but to the no notable difference of the intake of two kinds of polypeptides.
The intake of attached drawing 15, U87 Vitro Tumors spherical model to Fluorescein labeling polypeptides
Figure is what Fluorescein was markedDAE andLAE acts on the fluorescence tomography after 4h with U87 Vitro Tumor spherical models respectively Scan image, it is seen that U87 Vitro Tumor spherical models pairDAE andLThe intake of AE is apparently higher than free fluorescein, but to two kinds of polypeptides The no notable difference of intake.
Distribution in the lotus subcutaneous transplantation tumor nude mouse of Figure 16, Cy7 labeling polypeptide
Wherein, figure A is the Ex vivo Tumor image after lotus U87 subcutaneous transplantation tumor nude mice tail vein injection Cy7 labeling polypeptides 2h Distribution results；Scheme the fluorescence semi-quantitative results that B is Ex vivo Tumor；Scheme the fluorescence distribution image that C is Ex vivo Tumor and internal organs；Scheme D For the fluorescence semi-quantitative results of Ex vivo Tumor and internal organs, the results showed that, Cy7 labelsDAE andLThe accumulations of AE within the tumor are aobvious It writes higher than free Cy7 (* * * p<0.001),DAE cancer target effects are better thanLAE。
Intake of Figure 17, U87 Vitro Tumor spherical model to load C6 micellas
Figure isDAE-Micelle/C6、LAE-Micelle/C6, mPEG-Micelle/C6 and U87 Vitro Tumor spherical model are thin Born of the same parents act on the visible U87 tumours ball pair of fluorescence tomoscan image after 30minDAE-Micelle、LThe intake of AE-Micelle It is significantly higher than unmodified micella.
Figure 18, distribution in the subcutaneous tumors nude mouse of near infrared fluorescent dye micella is carried
Wherein, figure A is that tail vein injection carries near infrared fluorescent dye DiR micellas Each point in time in body fluorescence distribution figure Picture；Figure B is the fluorescence distribution image of Ex vivo Tumor and internal organs after giving preparation for 24 hours；Figure C is the fluorescence sxemiquantitative statistics knot for scheming B Fruit, the results showed that, mPEG-Micelle is compared,DAE-Micelle andLAE-Micelle can preferably targeted to tumor locus, AndDThe Targeting Effect of AE-Micelle is better thanLAE-Micelle。
Figure 19, the grain size for carrying taxol micella
Figure is each grain size for carrying taxol micella, and as seen from the figure, each prescription micellar size and form are without significant difference.
Figure 20, anti-U87 cells and HUVEC cell activity curves outside taxol glue bundle body are carried
Wherein, figure A and figure B be respectively mPEG-Micelle/PTX,DAE-Micelle/PTX、LAE-Micelle/PTX and The activity curve of taxol anti-U87 cells and HUVEC cells.After figure A shows U87 cell administrations 4h cultures 72h, IC50Respectively 0.21,0.02,0.05 and 0.31nM.Three kinds of micellas can inhibit the growth in vitro of U87 cells, whereinDAE-Micelle/PTX External activity isL2.5 times of AE-Micelle/PTX.Figure B cultivates 72h, IC after showing HUVEC cell administrations 4h50Respectively 0.70,0.06,0.21 and 0.85nM.Three kinds of micellas can inhibit the growth in vitro of HUVEC cells, whereinDAE-Micelle/ PTX external activities areL3.5 times of AE-Micelle/PTX.
Figure 21, inhibition of the taxol micella to new vessels external model is carried
Wherein, figure A be mPEG-Micelle/PTX,LAE-Micelle/PTX、DAE-Micelle/PTX andTo body The inhibition photo of outer neovascularization model, figure B is new vessels formation rate quantitative result, compared to mPEG-Micelle/PTX,DAE-Micelle/PTX andLAE-Micelle/PTX inhibits the formation of new vessels more notable, andDThe inhibition of AE-Micelle is imitated Fruit is better thanLAE-Micelle(*p<0.05, * * p<0.01).
Figure 22, inhibition of the taxol micella to mimicry blood vessel external model is carried
Wherein, figure A be mPEG-Micelle/PTX,LAE-Micelle/PTX、DAE-Micelle/PTX andTo body The inhibition photo of outer mimicry vascular pattern, figure B is the formation rate quantitative result of each group mimicry blood vessel structure, compared to mPEG- Micelle/PTX,DAE-Micelle/PTX andLAE-Micelle/PTX inhibits the formation of mimicry blood vessel more notable, andDAE- The inhibition of Micelle is better thanLAE-Micelle(*p<0.05, * * * p<0.001).
Figure 23, taxol micella inhibition subcutaneous transplantation tumor effect is carried
Wherein figure A is the curve that each group nude mouse tumor volume changes over time, compared with physiological saline group, each administration group pair Tumour growth has inhibiting effect.Compared to mPEG-Micelle/PTX,DAE-Micelle/PTX andLAE-Micelle/PTX Tumour growth can be significantly inhibited, andDThe antitumor drug effects of AE-Micelle/PTX are significantly better thanLAE-Micelle/PTX (n= 8, * * * p<0.001), figure B be nude mice is put to death take out tumor tissues after weigh and for statistical analysis, the results showed that each group tumor It is great small to be:DAE-Micelle /PTX<LAE-Micelle/PTX<MPEG-Micelle/PTX,DThe suppression of AE-Micelle/PTX Tumor most pronounced effects (n=8, * * * p<0.001).
Figure 24, TUNEL coloration result
Figure be mPEG-Micelle/PTX,LAE-Micelle/PTX、DAE-Micelle/PTX andSubcutaneous tumors TUNEL stained photographs (bar=100 μm), wherein nucleus are positive apoptotic cells in brown color or sepia, with mPEG- Micelle/PTX is compared,DAE-Micelle/PTX andLAE-Micelle/PTX promotes tumor tissues apoptosis more notable, andDAE- The apoptosis-promoting effect of Micelle is better thanLAE-Micelle。
The bis- coloration results of Figure 25, CD31/PAS
Figure be mPEG-Micelle/PTX,LAE-Micelle/PTX、DAE-Micelle/PTX andCD31/ The bis- stained photographs of PAS (bar=100 μm), nucleus is new vessels in brown color or sepia, with mPEG-Micelle/ PTX is compared,DAE-Micelle/PTX andLAE-Micelle/PTX inhibits the formation of new vessels more notable, andDAE-Micelle New vessels inhibiting effect be better thanLAE-Micelle。
Specific implementation mode
It will be helpful to further understand the present invention by following embodiments, but the present invention is not limited to range described below.
Embodiment 1
AE、AE-Fluorescein、AE-Cy7、AE-DTPA-Gd、AE-DTPA-99mTc, AE- drug, AE-PEG-PLA Synthesis and characterization
1)LAE andDAE、LAE-Cys andDThe synthesis of AE-Cys and characterization
Using solid phase polypeptide synthesis, designs and synthesizes and be made of D amino acidsD(sequence is AEDFDADLDGDEDA) andD(sequence is AE-CysDFDADLDGDEDADC) and L-configuration amino acid is constitutedL(sequence is AE FALGEA) andLAE-Cys (sequence FALGEAC).
Specific method：With Boc solid phase polypeptide synthesis, sequentially row are sequentially ingressed into amino acid on PAM resins, with HBTU/ DIEA is condensing agent, TFA is that deprotection agent is reacted.After the completion of reaction, resin is carried out with the hydrogen fluoride containing P-cresol Cutting, ice bath stirring react 1h, and decompression after reaction pumps hydrogen fluoride, and ice ether precipitate and washs precipitation 3 times, precipitate with 20% acetonitrile re-dissolves, and is rotated after collecting filtrate, obtains polypeptide crude product solution, and polypeptide crude product (contains 0.1% with acetonitrile/water TFA) system isolates and purifies.HPLC and ESI-MS characterizationsDAE、DAE-Cys、LAE andLThe purity and molecular weight (Mw) of AE-Cys, HPLC collection of illustrative plates, mass spectrogram are as shown in Figure 1, Figure 2, Figure 3 and Figure 4；
2) synthesis of AE-Fluorescein and AE-Cy7 and characterization
It will be obtained aboveDAE-Cys orLAE-Cys is dissolved in the PBS solution of 0.1M (pH7.2), Fluorescein-5- Maleimide is dissolved in DMF, and magnetic agitation is reacted after the two mixing, and HPLC monitorings prepare liquid phase purifying, use after complete reaction System was isolated and purified, was freeze-dried acetonitrile/water (containing 0.1%TFA)DAE-Fluorescein orLAE-Fluorescein is pure Product, HPLC collection of illustrative plates, mass spectrogram are as shown in Figure 5,6；
The preparation method of AE-Cy7 is same as above, and HPLC collection of illustrative plates, mass spectrogram are as shown in Figure 7,8；
3) AE-DTPA-Gd and AE-DTPA-99mThe preparation of Tc
Maleimide-DTPA is dissolved in DMF, ibid withDAE-Cys orLReaction is mixed in the PBS solution of AE-Cys, prepares Liquid phase purifies, and is freeze-driedDAE-DTPA orLAE-DTPA sterlings, chelate Gd or99mTc is up to AE-DTPA-Gd or AE- DTPA-99mTc；
4) preparation of AE- medicinal compositions, including,
It is prepared using AE- adriamycin composites as example of the AE connections containing ketone or aldehyde radical drug；9.4mg AE-Cys(DAE- Cys orLAE-Cys 3mL phosphate buffers (0.1mM, pH 7.0)) are dissolved in, three (2- carboxyethyls) phosphines of 10 times of moles are added (TCEP), 20min is stirred in 4 DEG C.Then the own hydrazine derivate of adriamycin 6- maleimides of 4 times of moles is added, in room temperature It is protected from light 1h.Reaction solution is purified with liquid phase is prepared, and is freeze-driedDAE orLAE- adriamycin composites.
The example of hydroxyl or amino drug is connected with disulfide bond using AE- taxols compound as AE；200mg Japanese yews Alcohol is dissolved in 10mL chloroforms, is cooled to 0-5 DEG C, and 39.99mg DCC and 60.4mg 3- (2- pyridines dimercapto) third is successively added Acid, after charging, is warmed to room temperature reaction overnight, and reaction solution filtering purifies (CHCl through column chromatography3/ MeOH=50:1-15:1, V/V is eluted) taxol 3- (2- pyridines dimercapto) propanoic derivatives are obtained, taxol 3- (2- pyridines dimercapto) propanoic derivatives are molten In 5mL DMF, the AE-Cys of 1.5 times of moles is dissolved in PBS/DMF solution, and solution ph keeps 4~5, by taxol 3- (2- pyridines dimercapto) propanoic derivatives are added dropwise in sulfydryl polypeptide solution, in room temperature reaction 6h, through preparing liquid phase purifying freeze-drying Obtain AE- taxol compounds；
Using AE- boron for assistant miaow compound as the example of AE connection boracic acid groups drugs, according to the synthesis of AE in resin On be sequentially ingressed into amino acid, wait for polypeptide all amino acid residues access finish, trifluoroacetic acid slough nitrogen end Boc protection.Add The DMF solution for entering succinic anhydride and DIEA containing 3 times of moles, in room temperature reaction 30min.After washing resin, it is added 5 times moles The trim,ethylchlorosilane of amount protects dopamine, and using HBTU/DIEA as condensing agent, in room temperature reaction 1h.Resin is cut with HF, And AE- DOPA amine derivatives are purified to obtain through preparative HPLC, in the buffer solution of pH7.4, AE- DOPA amine derivative is with boron for assistant Miaow is with molar ratio 1:1 mixes up to AE- boron for assistant miaow compound；
Using AE-PMI fused polypeptides as the example of AE connecting peptides drugs, directly it is made by solid phase polypeptide synthesis, Specific method is：After determining AE-PMI polypeptide sequences, it is sequentially ingressed into amino acid by method identical with AE is prepared, simultaneously through HF cuttings AE-PMI fused polypeptides are obtained after purification；
5) synthesis of AE-PEG-DSPE and characterization
It willDAE-Cys orLAE-Cys is dissolved in the PBS solution of 0.1M (pH7.2), and Mal-PEG-PLA is taken to be dissolved in DMF, and two Magnetic agitation is reacted after person's mixing, and HPLC monitorings stop reaction after the reaction was complete after Mal-PEG-DSPE, excessiveDAE-Cys、LAE-Cys and DMF dialysis (molecular cut off 3.5kDa) removes, and is freeze-driedDAE-PEG-DSPE orLAE-PEG-DSPE, NMR characterizes its structure (as shown in Figure 9)；
6) synthesis of AE-PEG-PLA and characterization
It willDAE-Cys orLAE-Cys is dissolved in the PBS solution of 0.1M (pH7.2), and Mal-PEG-PLA is taken to be dissolved in DMF, and two Magnetic agitation is reacted after person's mixing, and HPLC monitorings stop reaction after the reaction was complete after Mal-PEG-PLA, excessiveDAE-Cys、LAE-Cys and DMF dialysis (molecular cut off 3.5kDa) removes, and is freeze-driedDAE-PEG-PLA orLAE-PEG-PLA, NMR Characterize its structure (as shown in Figure 10).
The preparation of 2 AE liposomes of embodiment/Dox
PEG- liposome membrane material compositions are HSPC/Chol/mPEG2000-DSPE(52:43:5, mol/mol), more The PEG liposome membrane material prescriptions of peptide modification are HSPC/Chol/mPEG2000- DSPE/ polypeptides-PEG-DSPE (52:43:3:2, Mol/mol), weigh above-mentioned membrane material and be dissolved in chloroform, decompression rotary evaporation removes organic solvent, obtains uniform lipid film, and vacuum is dry It is dry to be prepared using ammonium sulphate gradient for 24 hours and contain each liposome of adriamycin (DOX), dynamic light scattering determination particle diameter distribution (as shown in figure 11).
The serum stability of 3 AE of embodiment is investigated
It willDAE andLAE is made into 1mg/mL aqueous solutions, and 0.1mL is taken to be added in 50% mice serum of 0.9mL, 37 DEG C of incubations, 100 μ L are taken out respectively at 0,5 and 15min, 0.5,1,2,4,6 and 8h, egg in 20 μ L trichloroacetic acids (TCA) precipitation serum is added In vain, 4 DEG C of standings 20min, 12000 revs/min of centrifugation 10min take 20 μ L of supernatant to carry out HPLC analyses, serum stability knot Fruit (as shown in figure 12) showsDAE have thanLThe better serum stabilities of AE.
The cell in vitro targeting of 4 AE of embodiment is verified
1) targetings of the AE to brain glioblastoma cell U87
The brain glioblastoma cell (U87 cells) of the monolayer cultivation of logarithmic growth phase, with 0.25% trypsin digestion list Layer culture cell, is made into single cell suspension, with every hole 1 × 10 with the DMEM culture solutions containing 10% fetal calf serum5A cell inoculation In 12 well culture plates, per pore volume 1mL, culture plate is moved into carbon dioxide incubator, 37 DEG C, 5%CO2And saturated humidity Under the conditions of cultivate for 24 hours, with the DMEM culture solution compound concentrations containing 10% fetal calf serum be 5 μM FAM,DAE-Fluorescein AndLCulture solution in culture plate is sucked out AE-Fluorescein solution, is separately added into above-mentioned solution, 37 DEG C of incubation 4h, and suction is abandoned Supernatant is washed three times with PBS solution, 4% paraformaldehyde fixer fixation cell, after DAPI nuclear targetings, laser co-focusing Observation, cell internalizing photo is as shown in Figure 13 left figures, after separately being washed three times with PBS, flow cytometry analysis is carried out, as a result such as Figure 13 Shown in right figure；
2) targetings of the AE to Human umbilical vein endothelial cells HUVEC
The Human umbilical vein endothelial cells (HUVEC cells) of the monolayer cultivation of logarithmic growth phase, are ibid tested, cell internalizing Photo is as shown in Figure 14 left figures, and flow cytometry analysis result is as shown in Figure 14 right figures；
3) targetings of the AE to U87 Vitro Tumor spherical models
It takes 48 well culture plates to be added per hole under 150 μ L, UV light of Agrose sol solutions and irradiates 30min after its solidification, 400 μ L U87 cell suspensions are inoculated with per hole, cell density is 2 × 103A/hole, is placed in carbon dioxide incubator, 37 DEG C, 5% CO2And cultivated under the conditions of saturated humidity 7 days and form tumour ball, it is with the DMEM culture solution compound concentrations containing 10% fetal calf serum 5 μM of FITC,DAE-Fluorescein andLCulture solution in culture plate is sucked out, is separately added by AE-Fluorescein solution Above-mentioned solution, 37 DEG C of incubation 4h, suction is abandoned supernatant, is washed three times with PBS solution, and after paraformaldehyde is fixed, fluorescence microscope is seen It examines, photo is as shown in figure 15.
5 AE in-vivo tumour targetings of embodiment are verified
Subcutaneous tumors animal model is built first, by the U87 cell tryptase enzymic digestions in exponential phase, adjusts cell concentration It is 3 × 107A/mL, 100 μ L of inoculation are subcutaneous to the nearly armpit of the right back side of nude mouse, and in SPF grades, routine observation is swollen for raising after inoculation Tumor size waits for that tumor size is 200mm3When, the tumor bearing nude mice without necrosis, tumor shape rule is filtered out, grouping is tested, The Cy7 of the identical fluorescence intensities of 200 μ L,DAE-Cy7 andLAE-Cy7 solution is injected by tail vein in tumor bearing nude mice animal model body, It puts to death nude mice after 2h, takes out tumour, with distribution of the living imaging instrument detection fluorescence in tumor bearing nude mice body and carry out fluorescence semidefinite Amount calculates, as a result as shown in figure 16.
6 AE-Micelle targetings of embodiment are verified
1) preparation of AE-Micelle/C6 micellas
The prescription of AE-Micelle is 9mg mPEG-PLA, 1mg AE-PEG-PLA, weighs above-mentioned micellar material and 5ug is fragrant Legumin -6 is dissolved in 2ml acetonitriles, 37 DEG C of water-baths, and decompression (~0.085MPa) rotary evaporation removes organic solvent, at uniform films, Room temperature in vacuo is dried overnight, and 2ml physiological saline aquations are added, and CL-4B column chromatographies remove non-encapsulated C6, and AE- is made Micelle/C6；
2) targetings of the AE-Micelle to U87 Vitro Tumor spherical models
With the DMEM culture solutions containing 10% fetal calf serum prepare a concentration of 5 μM of mPEG-Micelle/C6 of C6,DAE- Micelle/C6 andLThe culture solution being built in the culture plate of tumour ball is sucked out, is separately added by AE-Micelle/C6 solution Above-mentioned solution, 37 DEG C of incubation 30min, suction is abandoned supernatant, is washed three times with PBS solution, after paraformaldehyde is fixed, fluorescence microscope Observation, as a result as shown in figure 17.
Targeting is verified in 7 AE-Micelle bodies of embodiment
1) preparation of AE-Micelle/DiR
AE-Micelle/DiR is prepared to prepare the identical methods of AE-Micelle/C6；
2) targeting is verified in AE-Micelle bodies
The mPEG-Micelle/DiR of 100 μ L of difference tail vein injection,DAE-Micelle/DiR andLAE-Micelle/DiR Solution, respectively after injection 4,6,8,12 and for 24 hours when anesthetized mice, with living imaging instrument record fluorescence in tumor bearing nude mice body It is distributed and carries out fluorescence sxemiquantitative calculating, as a result as shown in figure 18.
8 AE-Micelle/PTX Pharmacodynamics in vitro of embodiment is tested
1) preparation of AE-Micelle/PTX and characterization
AE-Micelle/PTX is prepared to prepare the identical methods of AE-Micelle/C6.Laser diffraction particle size instrument levies glue Beam grain size and particle diameter distribution (as shown in figure 19)；
2) AE-Micelle/PTX pharmacy in vitro is tested
With 4.0 × 103A/hole by U87 cells or HUVEC cell inoculations in 96 orifice plates, for 24 hours after, culture solution is sucked out, is added Enter a series of 200 concentration of μ L mPEG-Micelle/PTX,DAE-Micelle/PTX、LAE-Micelle/PTX and taxol, administration After 4h cultivates 72h, MTT solution is added and continues to cultivate 4h, discards culture solution, 150 μ L DMSO are added, oscillation is molten to purple particle Solution, absorbance value is measured with microplate reader at 590nm, and cell survival rate is measured using mtt assay, calculates half lethal dose, knot Fruit is as described in Figure 20；
3) AE-Micelle/PTX tests the inhibition that new vessels are formed
It takes 24 well culture plates that 50 μ L matrigels are added per hole, is laid in 24 orifice plates, 30min is incubated in 37 DEG C of incubators and is waited for It is solidified, and 0.25% pancreatin digests HUVEC cells, with the DMEM cultures of micella or free paclitaxel liquid containing 1 μM of taxol Liquid is made into single cell suspension, with every hole 1 × 105A cell inoculation is in 24 well culture plates, 37 DEG C, 5%CO2And saturated humidity item Observation capillary structure forms (as illustrated in fig. 21) and calculates formation rate (such as Figure 21 B of capillary structure after cultivating 12h under part It is shown)；
4) AE-Micelle/PTX tests the inhibition of mimicry vascularization
It takes 24 well culture plates that 50 μ L matrigels are added per hole, is laid in 24 orifice plates, 30min is incubated in 37 DEG C of incubators and is waited for It is solidified, and 0.25% pancreatin digests U87 cells, with the DMEM culture solutions of micella or free paclitaxel liquid containing 1 μM of taxol It is made into single cell suspension, with every hole 1 × 105A cell inoculation is in 24 well culture plates, 37 DEG C, 5%CO2And saturated humidity condition Observation capillary structure forms (as shown in fig. 22) and calculates formation rate (such as Figure 22 B of mimicry blood vessel structure after lower culture 12h It is shown).
The test of pesticide effectiveness in 9 AE-Micelle/PTX bodies of embodiment
1) AE-Micelle/PTX inhibits to test to subcutaneous transplantation tumor
U87 subcutaneous tumors animal models are built, routine observation tumor size waits for that tumor size is 100mm3When, grouping carries out Experiment, difference tail vein injection mPEG-Micelle/PTX,DAE-Micelle/PTX、LAE-Micelle/PTX, commercially available taxol with And each 100 μ l of physiological saline, the total dosages of PTX of administration group are 25mg/kg, are divided into five times, and each dosing interval is two days, Every other day with the major diameter (a) and minor axis (b) of vernier caliper measurement tumour, each group nude mouse tumor volume is calculated according to formula, is drawn swollen Knurl accumulates versus time curve, calculates each group significant difference,
Gross tumor volume calculation formula：VKnurl is accumulated=0.5 (a × b2)
After administration 15 days (after inoculation 21 days), the neck that breaks puts to death all nude mices, takes subcutaneous tumor and weighs, and calculates each group system Meter learns difference (as shown in figure 23)；
2) AE-Micelle/PTX promotees apoptosis experiment
After tumor bearing nude mice completes five administrations, puts to death taking-up tumor tissue and be fixed, specimens paraffin embedding slices.It is de- using end DUTP Nick Ends labelling method (the Terminal deoxynucleotidyl that oxygen nucleotidyl transferase (TDT) mediates Transferase-mediated dUTP nick end labeling, TUNEL) detection tumour cell apoptosis degree, cell Core is determined as apoptotic cell in brown color or sepia, and 3 high power fields are observed continuously under an optical microscope and count the positive Cell, as a result as shown in figure 24；
3) AE-Micelle/PTX inhibits to test to tumor vessel
After tumor bearing nude mice completes five administrations, puts to death taking-up subcutaneous tumors and fix, it is immune to carry out CD31 for specimens paraffin embedding slices Histochemical staining and the bis- dyes of PAS, observe angiogenesis situation, as a result as shown in figure 25 under an optical microscope.

Claims (17)

1.AE polypeptides, which is characterized in that it includes D configuration polypeptidesDAE, wherein D amino acids sequences areDFDADLDGDEDA, and L-configuration polypeptideLAE, wherein L-configuration amino acid sequence is FALGEA.
2. AE polypeptides described in claim 1, which is characterized in that D configurations polypeptide thereinDAE 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 polypeptidesDAE 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 polypeptidesDAE 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 ofDAE-X compounds, which is characterized in that use D configurations polypeptide described in claim 1DAfter AE sulfhydrylations with contain The image substance reaction of maleimide base group obtains.
6. a kind ofLAE-X compounds, which is characterized in that using described in claim 1LAfter AE sulfhydrylations and contain maleimide The image substance reaction of amine groups obtains；DescribedLAE-X compounds while targeting epidermal growth factor receptor and epidermal growth Factor acceptor mutant III.
7. by described in claim 5 or 6DAE-X compounds orLAE-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 Agent99mTc-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 thereinDHydrazone 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, obtainsDAE- Y compounds；
L-configuration polypeptide thereinLAE 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 obtainsLAE-Y compounds.
9. AE polypeptides as described in claim 9, which is characterized in that describedDAE-Y compounds orLY 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 thereinDAfter AE sulfhydrylations and contain Malaysia Polyethylene glycol-Z the compounds of imide group connect, and obtainDAE- polyethylene glycol-Z compounds；
It is thereinLIt connect, obtains with the polyethylene glycol-Z compounds of maleimation after AE sulfhydrylationsLAE- polyethylene glycol-Z is multiple Close object.
11. AE polypeptides as described in claim 10, which is characterized in that describedDAE- polyethylene glycol-Z compounds or andLAE- 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 obtainedDAE- polyethylene glycol-phosphorus fat complexes OrLAE- 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 obtainedDAE- 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 orLAE- 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.
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