WO2017063542A1 - 稳定化a7r多肽及其在构建肿瘤靶向诊治递药系统中的用途 - Google Patents

稳定化a7r多肽及其在构建肿瘤靶向诊治递药系统中的用途 Download PDF

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WO2017063542A1
WO2017063542A1 PCT/CN2016/101738 CN2016101738W WO2017063542A1 WO 2017063542 A1 WO2017063542 A1 WO 2017063542A1 CN 2016101738 W CN2016101738 W CN 2016101738W WO 2017063542 A1 WO2017063542 A1 WO 2017063542A1
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tumor
polypeptide
stabilized
drug
group
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PCT/CN2016/101738
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English (en)
French (fr)
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陆伟跃
应曼
谢操
高洁
宋现飞
李雪
张明菲
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复旦大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links

Definitions

  • the invention belongs to the field of pharmacy, and relates to a reverse-order D-configuration polypeptide and a head-tail amide bond cyclized polypeptide which are highly stable and can simultaneously target vascular endothelial growth factor receptor 2 and neuropilin-1 high expression cells, and the drug complex and modification thereof
  • the nano drug delivery system and the combination with an antitumor drug specifically related to the D configuration polypeptide D A7R (D configuration D amino acid sequence D R D P D P D L D W D T D A), amide bond cyclization polypeptide cA7R ( L-configuration amino acid sequence c (CATWLPPR)), its diagnostic and therapeutic drug complexes, modified polymer carrier materials, liposome, polymer micelles and other nano-delivery systems, as well as in tumor diagnosis and tumor targeting Application in therapy; D A7R, cA7R synergistic anti-tumor drug application.
  • Tumors are diseases that pose a serious threat to human life and health, and mortality is the highest among all disease deaths.
  • Traditional chemotherapy as the main means of cancer drug treatment, has the defects of poor selectivity, high toxicity, narrow therapeutic window and easy multidrug resistance.
  • nano-drug delivery systems have received more and more attention in recent years.
  • the nano-dose delivery system has the advantages of high drug loading and long circulation time in the body. It can utilize the EPR effect of the tumor to enable the drug to be passively enriched in the tumor site, but the efficiency is low.
  • the active targeting strategy is directed to receptors or transporters that are highly expressed in tumor tissues, and the nano-delivery system is delivered to tumor tissues or cells using corresponding ligands that have the ability to recognize and bind to specific receptors or transporters.
  • corresponding ligands include monoclonal antibodies, polypeptides, nucleic acid aptamers, small molecule compounds, and the like.
  • the ligand-modified nano-delivery system can be enriched in the tumor site by EPR effect, and then the drug can be delivered to tumor tissues and cells through specific recognition, binding and internalization of cell surface receptors or transporters and ligands. In order to achieve the active targeting target of the nano drug delivery system for tumors.
  • Vascular endothelial growth factor receptor 2 (VEGFR2) is a specific receptor for vascular endothelial growth factor (VEGF), which is mainly expressed in vascular endothelial cells and most tumor cells.
  • VEGF/VEGFR2 signaling pathway is the most important rate-limiting step in physiological and pathological angiogenesis and is critical for tumor angiogenesis.
  • VEGF and VEGFR2 are not only the most important targets for various anti-tumor vascular targeted therapies in clinical practice, but also a hot spot in the basic research field of oncology.
  • Neuropilin-1 (NRP-1) is a transmembrane glycoprotein that is a co-receptor of Sema3A and VEGF165 and plays an important role in tumor angiogenesis, tumor growth and metastasis. Studies have shown that NRP-1 is not only expressed in tumor vascular endothelial cells, but also overexpressed on a variety of tumor cell membranes, including glioma, lung cancer, pancreatic cancer, prostate cancer, breast cancer and melanoma. In addition, the dysfunction of glioma blood vessels and high gap pressure limit the penetration of drugs into vascular endothelial cells into the tumor parenchyma, resulting in a decrease in the efficacy of the drug.
  • CendR polypeptide (a series of peptides composed of R/KXXR/K amino acid sequences) binds to NRP-1 receptor and increases the permeability of blood vessels and tumor tissues, and enhances the penetration of drugs or therapeutic genes into deep tissues of tumors. ability.
  • Phage display technology is one of the important means to screen effective ligands for targeting tumor tissues.
  • the peptides screened by this technology can be applied to tumor diagnosis and treatment.
  • L A7R (L-configuration amino acid sequence ATWLPPR) is a heptapeptide that is highly conjugated to VEGFR2 and NRP-1 by phage display technology, and can target tumor neovascularization, mimetic blood vessels and high expression of VEGFR2 and NRP-1. Tumor cells; however, L A7R is less stable in vivo and is easily degraded in the blood, thereby reducing its tumor targeting ability.
  • the present invention provides a stabilized A7R polypeptide and its use in tumor targeted diagnosis and utilization of synergistic anti-tumor drugs, in particular, in the preparation of a high stability A7R reverse order D
  • the configuration polypeptide D A7R (D configuration amino acid sequence D R D P D P D L D W D T D A) and the head-tail amide bond cyclization polypeptide cA7R (L configuration amino acid sequence c (CATWLPPR)), and used to modify the drug Molecular and polymeric carrier materials, the stability of A7R drug complexes, the stability of the drug-loaded A7R-nano drug delivery system, to improve the targeted diagnosis and treatment of drugs; stability A7R itself has anti-tumor effect, further The combination of anti-tumor drugs has synergistic effects.
  • the present invention provides a stabilized A7R polypeptide which is a reverse D-configuration polypeptide D A7R and/or a head-to-tail amide bond cyclization polypeptide cA7R, wherein the amino acid sequence of the reverse D-configuration polypeptide D A7R is D R D P D P D L D W D T D A, the L-configuration amino acid sequence of the head-tail amide bond cyclized polypeptide cA7R is c (CATWLPPR). Wherein the amino acid sequence of cA7R is as shown in SEQ ID NO: 1.
  • the reverse D-configuration polypeptide D A7R was designed and prepared, and the head-tail amide bond cyclized polypeptide cA7R was designed and prepared according to the “Native Chemical Ligation” reaction, and both polypeptides have high serum. It has high affinity with vascular endothelial growth factor receptor 2 (VEGFR2) and neuropilin-1 (NRP-1).
  • VAGFR2 vascular endothelial growth factor receptor 2
  • NPP-1 neuropilin-1
  • the present invention provides a stabilized A7R polypeptide complex, wherein the stabilized A7R polypeptide is modified to contain a maleimide group-containing imaging material, wherein The structure of the stabilized A7R polypeptide complex is D A7R-X and/or cA7R-X, and X is the image substance.
  • the X is selected from one or more of a fluorescent substance, a near-infrared dye, and a magnetic resonance imaging agent, and more preferably, the fluorescent substance is 5-carboxyfluorescein, and the near-infrared dye is selected from cy5. .5. One or more of IR820 and DiR, the magnetic resonance imaging agent is Gd-DTPA.
  • the cA7R designed by the present invention and the D A7R linked to cysteine can utilize a thiol group and a maleimide functionalized fluorescent substance (FAM) and a near-infrared dye (Cy5.5, IR820, DiR, etc. reacts with a magnetic resonance imaging agent (Gd-DTPA,) to form a complex.
  • FAM maleimide functionalized fluorescent substance
  • Gd-DTPA magnetic resonance imaging agent
  • the present invention provides a method for producing the aforementioned stabilized A7R polypeptide complex, which comprises the aforementioned stabilized A7R polypeptide or the thiolated aforementioned stabilized A7R polypeptide and The image substance having a maleimide group reacts.
  • the present invention provides a stabilized A7R polypeptide complex, which is the aforementioned stabilized A7R polypeptide modified antitumor drug, wherein the structure of the stabilized A7R polypeptide complex is D A7R-Y and/or cA7R-Y, Y are the antitumor drugs.
  • the antitumor drug is selected from one or more of a ketone or aldehyde group-containing drug, a hydroxyl group- or amino group-containing drug, a boric acid group-containing drug, and a polypeptide drug.
  • the ketone- or aldehyde-based drug is doxorubicin or epirubicin
  • the hydroxy- or amino-containing drug is selected from the group consisting of paclitaxel, docetaxel, camptothecin, hydroxycamptothecin, 9- Nitrocamptothecin, vincristine, etoposide, gemcitabine, cytarabine, 5-fluorouracil, teniposide, moritinib, epothilone, vinorelbine, actinomycin D, rice Toxic, mitomycin, bleomycin, irinotecan, the boronic acid group-containing drug is bortezomib or carfilzomib, and/or the polypeptide drug is selected from the group consisting of p53 activating peptides, bees One or more of a toxic peptide and a muscarinic peptide.
  • the cA7R and D A7R modified drugs designed by the present invention comprise a pH-sensitive oxime bond formed by reacting a maleimide hexanthene derivative (involving a ketone- or aldehyde-containing drug such as doxorubicin or epirubicin).
  • the present invention provides a method of preparing the aforementioned stabilized A7R polypeptide complex, the method comprising:
  • the stabilized A7R polypeptide is prepared by linking the anti-tumor drug with a pH-sensitive sputum bond;
  • the antitumor drug is a drug containing a hydroxyl group or an amino group
  • the aforementioned stabilized A7R polypeptide is linked to the antitumor drug through a disulfide bond to prepare the stabilized A7R polypeptide complex
  • the antitumor drug is a boric acid group-containing drug
  • the aforementioned stabilized A7R polypeptide is linked to the antitumor drug by a pH-sensitive borate bond to prepare the stabilized A7R polypeptide complex
  • the antitumor drug is a polypeptide drug
  • the stabilized A7R polypeptide is condensed with the antitumor drug to prepare the stabilized A7R polypeptide complex.
  • the present invention provides a stabilized A7R polypeptide complex, wherein the stabilized A7R polypeptide complex is the aforementioned stabilized A7R polypeptide modified polymer carrier material, wherein the structure of the stabilized A7R polypeptide complex It is D A7R-polyethylene glycol-Z and/or cA7R-polyethylene glycol-Z, Z is the polymer carrier material.
  • the polymeric carrier material is selected from one or more of phospholipids, polylactic acid, lactic acid glycolic acid copolymers, and polycaprolactones.
  • the cA7R designed by the present invention and the D A7R linked to cysteine can be modified in a polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) containing a maleimide functional group, Polyethylene glycol-polylactic acid (PEG-PLA), polyethylene glycol-lactic acid glycol copolymer (PEG-PLGA), polyethylene glycol-polycaprolactone (PEG-PCL) and other polymer carrier materials, It can be used for the construction of D A7R or cA7R modified liposome, polymer micelle, polymer disc, nanoparticle and other nano drug delivery systems.
  • PEG-DSPE polyethylene glycol-distearoylphosphatidylethanolamine
  • PEG-DSPE polyethylene glycol-distearoylphosphatidylethanolamine
  • PEG-DSPE polyethylene glycol-distearoylphosphatidylethanolamine
  • PEG-DSPE polyethylene glycol-d
  • the present invention provides a method for preparing the aforementioned stabilized A7R polypeptide complex, comprising: the aforementioned stabilized A7R polypeptide or the thiolated aforementioned stabilized A7R polypeptide and maleimide-polyethylene
  • the stabilized A7R polypeptide complex is prepared by reacting a diol-polymer carrier material.
  • the invention provides a delivery system comprising the aforementioned stabilized A7R polypeptide complex.
  • the delivery system is a liposome delivery system, a polymeric micellar delivery system, a polymer disc delivery system, or a nanoparticle delivery system.
  • the present invention also provides the aforementioned delivery system further comprising (1) an imaging substance and/or (2) an anti-tumor drug other than the stabilized A7R polypeptide complex.
  • the (1) imaging substance is selected from one or more of a fluorescent substance, a near-infrared dye, and a magnetic resonance imaging agent. More preferably, the fluorescent substance is 5-carboxyfluorescein (5-FAM), and the near-infrared dye is selected from one or more of Cy5.5, IR820, DiR and DiD, and/or magnetic resonance image
  • the agent is Gd-DTPA.
  • the (2) antitumor drug is selected from the group consisting of doxorubicin, epirubicin, paclitaxel, docetaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, vincristine, boron Tezomib, carfilzomib, cyclophosphamide, etoposide, gemcitabine, cytarabine, 5-fluorouracil, teniposide, moritinib, epothilone, vinorelbine, actinomycin D , mitoxantrone, mitomycin, bleomycin, irinotecan, cisplatin, oxaliplatin, p53 activating peptide, melittin, scorpion venom, bevacizumab and trastuzumab One or more.
  • the D A7R or cA7R modified nano-delivery system designed by the present invention may contain doxorubicin, epirubicin, paclitaxel, docetaxel, camptothecin, hydroxycamptothecin, 9-nitro-Hi Alkaloid, vincristine, bortezomib, carfilzomib, cyclophosphamide, etoposide, gemcitabine, cytarabine, 5-fluorouracil, teniposide, morifinib, epothilone, vinca Rebine, actinomycin D, mitoxantrone, mitomycin, bleomycin, irinotecan, cisplatin, oxaliplatin, p53 activating peptide, melittin, scorpion venom, bevac Monoclonal antibody, trastuzumab, etc.; can also contain fluorescent substances, near-infrared dyes and magnetic resonance imaging agents, such as FAM
  • the present invention provides the use of the aforementioned stabilized A7R polypeptide, the aforementioned stabilized A7R polypeptide complex, and the aforementioned delivery system for the preparation of a medicament or medical product for diagnosis, tracing and/or treatment of a tumor. .
  • the tumor is a high expression of a neovascular endothelial growth factor receptor 2 tumor or a high expression neuropilin-1 tumor.
  • the D A7R or cA7R designed by the present invention has an anti-tumor effect, and can be further administered in combination with an anti-tumor drug, such as doxorubicin, epirubicin, paclitaxel, docetaxel, camptothecin.
  • an anti-tumor drug such as doxorubicin, epirubicin, paclitaxel, docetaxel, camptothecin.
  • the D A7R and cA7R designed by the present invention can mediate drugs or nano drug delivery systems targeting cells and tissues thereof with high expression of vascular endothelial growth factor receptor 2 (VEGFR2) and neuropilin-1 (NRP-1).
  • VEGFR2 vascular endothelial growth factor receptor 2
  • NRP-1 neuropilin-1
  • the present invention provides a combination for diagnosing, tracing, and/or treating a tumor, the combination comprising one or more components selected from the group consisting of the aforementioned stabilized A7R polypeptide, the foregoing Stabilizing the A7R polypeptide complex and the aforementioned delivery system.
  • the combination product is a kit, and/or
  • the tumor is a high expression of a neovascular endothelial growth factor receptor 2 tumor or a high expression neuropilin-1 tumor.
  • the present invention provides a method of diagnosing, tracing, and/or treating a tumor comprising administering an effective dose of an oral or parenteral route to a patient having the tumor or suspected of having the tumor.
  • a stabilized A7R polypeptide as described above a stabilized A7R polypeptide complex as described above, a delivery system as described above, and a combination of the foregoing.
  • the tumor is a high expression of a neovascular endothelial growth factor receptor 2 tumor or a high expression neuropilin-1 tumor; and/or
  • the method comprises administering an effective dose of (1) the aforementioned stabilized A7R to a patient having the tumor or suspected of having the tumor by an oral or non-oral route.
  • a polypeptide and (2) one or more other anti-tumor drugs preferably selected from the group consisting of: doxorubicin, epirubicin, paclitaxel, docetaxel, camptothecin, hydroxycamptothecin, 9 -nitrocamptothecin, vincristine, bortezomib, carfilzomib, cyclophosphamide, temozolomide, etoposide, guanidine, gemcitabine, cytarabine, 5-fluorouracil, teniposide, mo Litinib, epothilone, vinorelbine, actinomycin D, mitoxantrone, mitomycin, bleomycin, irinotecan, cisplatin
  • the oral or parenteral route can be delivered to the patient by oral, injection, patch, spray, and other known one or more.
  • the effective amount can include an amount effective to treat, reduce, alleviate, alleviate, eliminate, or condition one or more symptoms, the condition seeking to be treated, or alternatively, the condition seeking to be avoided, or otherwise A clinically identifiable favorable change is produced in the condition or its effect.
  • the present invention also provides the use of the aforementioned stabilized A7R polypeptide as an antitumor active ingredient in the preparation of a medicament for antitumor and/or a medical product. .
  • the invention also provides the use of the aforementioned stabilized A7R polypeptide for the preparation of a tumor targeting product.
  • the tumor targeting product is used to target tumor neovascularization, mimetic blood vessels and tumor cells with high expression of VEGFR2 and NRP-1; and/or the tumor targeting product is for diagnosis, tracing and/or Drugs, laboratory reagents, and/or medical products that treat tumors.
  • the present invention provides the aforementioned stabilized A7R polypeptide for use in the preparation for synergistic increase Applications in medicines, laboratory reagents and/or medical products that are effective against other anti-tumor drugs;
  • the other anti-tumor drug is preferably selected from the group consisting of: doxorubicin, epirubicin, paclitaxel, docetaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, vincristine, boron Tezomib, carfilzomib, cyclophosphamide, temozolomide, etoposide, guanidine, gemcitabine, cytarabine, 5-fluorouracil, teniposide, moritinib, epothilone, vinorelbine, Actinomycin D, mitoxantrone, mitomycin, bleomycin, irinotecan, cisplatin, oxaliplatin, p53 activating peptide, melittin, scorpion venom, bevacizumab and One or more of trastuzumab.
  • the present invention provides a reverse-order D-configuration polypeptide and a head-tail amide bond cyclized polypeptide which are highly stable and can simultaneously target vascular endothelial growth factor receptor 2 and neuropilin-1 high expressing cells, and construct complexes and modified nanometers thereof.
  • the drug delivery system realizes the targeted diagnosis and treatment of tumors; the high binding of A7R polypeptide to VEGFR2 is used to inhibit tumor angiogenesis and achieve anti-tumor effect.
  • A7R polypeptide also has CendR polypeptide-like structure targeting NRP- 1, can increase the penetration of drugs on tumor tissue, and thus further synergistic with anti-tumor drugs can synergistically.
  • the present invention provides a material basis for the preparation and properties of D A7R, cA7R, and the above-described modified drug complexes and nano drug delivery systems for tumor diagnosis and treatment.
  • a specific implementation can be as follows:
  • D A7R and D A7R-Cys were prepared by solid phase synthesis.
  • D A7R-Fluorescein was synthesized by Michael addition reaction of maleimide group with mercapto group.
  • the structure was characterized by HPLC and MS.
  • cA7R was prepared according to the "Native Chemical Ligation" reaction. cA7R-Fluorescein was synthesized by Michael addition reaction of a maleimide group with a thiol group. The structure was characterized by HPLC and MS. 3. D A7R and cA7R stability and receptor affinity evaluation
  • D A7R and cA7R properties were determined from serum stability, binding ability to vascular endothelial growth factor receptor 2 (VEGFR2) and neuropilin-1 (NRP-1), and cellular uptake ability to express both receptors Investigation.
  • D A7R, cA7R and L A7R were each incubated with mouse serum at 37 ° C, and the concentration of the polypeptide was measured at different time points for stability comparison.
  • the binding ability of D A7R, cA7R and L A7R to the two receptor proteins was evaluated by surface plasmon resonance. Comparison
  • vascular endothelial growth factor receptor 2 and neuropilin-1 protein eg, umbilical vein endothelial cells HUVEC
  • model tumor cells eg, brain gel
  • cA7R and D A7R linked to cysteine react with the maleimide hexamidine derivative of the drug to form a polypeptide-drug complex containing a pH-sensitive oxime bond, wherein the drug involved includes doxorubicin, epirubicin A drug containing a ketone or an aldehyde group.
  • cA7R and D A7R linked to cysteine react with a 3-(2-pyridyldithio)propionic acid derivative of the drug to form a disulfide-containing polypeptide-drug complex, wherein the drug involved includes paclitaxel, polyene A drug containing a hydroxyl group or an amino group such as paclitaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, vincristine or the like.
  • cA7R and D A7R form a polypeptide-drug complex containing pH-sensitive borate by modifying dopamine and then reacting with a boronic acid group of the drug, wherein the drug involved includes a drug containing a boric acid group such as bortezomib.
  • cA7R and D A7R are directly condensed with a polypeptide drug by solid phase synthesis to form a fusion polypeptide, and the drugs involved include peptide drugs such as p53 activating peptide, antimicrobial peptide, and polypeptide toxin.
  • D A7R-adriamycin complex D A7R-Aldoxorubicin
  • L A7R-adriamycin complex L A7R-Aldoxorubicin
  • D A7R-Aldoxorubicin and L A7R-Aldoxorubicin were respectively incubated with 0.1 M phosphate buffer at different pHs at 37 ° C, and the concentration of A7R-Aldoxorubicin was measured at different time points for comparison of pH stability.
  • D A7R-Aldoxorubicin and L A7R-Aldoxorubicin were incubated with mouse serum at 37 ° C, respectively, and the concentration of A7R-Aldoxorubicin was measured at different time points for comparison of serum stability.
  • D A7R-Aldoxorubicin, L A7R-Aldoxorubicin, Aldoxorubicin and free doxorubicin were injected into the tail vein of nude mice bearing subcutaneous xenograft model. After 1 hour, the tumors were taken out to make frozen sections, CD31 labeled blood vessels, DAPI stained nucleus, and each group of drugs was compared. Distributed within the tumor.
  • the U7 subcutaneous xenograft model was injected into the tail vein of nude mice with D A7R-Aldoxorubicin, L A7R-Aldoxorubicin, Aldoxorubicin, free doxorubicin and normal saline.
  • the tumor anti-tumor effect was evaluated by tumor volume and tumor weight.
  • mice in each subcutaneous tumor pharmacodynamic test were dissected and fixed in 4% paraformaldehyde in PBS solution, and paraffin-embedded sections were subjected to HE staining to examine the systemic toxicity of each group of drugs.
  • the U7 subcutaneous xenograft model was injected into different doses of D A7R-Aldoxorubicin, free doxorubicin, peptide D A7R and normal saline.
  • the tumor anti-tumor effect was evaluated by tumor volume and tumor weight.
  • D A7R, cA7R, L A7R modified polymer materials D A7R-PEG-DSPE, cA7R-PEG-DSPE and L A7R-PEG-DSPE were synthesized.
  • D A7R-Cys was reacted with Mal-PEG-DSPE in a mixed solution of PBS and DMF at pH 7.2 to obtain D A7R-PEG-DSPE.
  • cA7R, L A7R-Cys were reacted with Mal-PEG-DSPE as described above to obtain cA7R-PEG-DSPE and L A7R-PEG-DSPE.
  • D A7R, cA7R, L A7R modified liposomes ( D A7R-PEG-liposome, cA7R-PEG-liposome and L A7R-PEG-liposome) were then prepared separately.
  • Liposomes were prepared by membrane-forming hydration method with a certain proportion of HSPC/Chol/mPEG 2000 -DSPE/ D A7R-PEG-DSPE or cA7R-PEG-DSPE or L A7R-PEG-DSPE as membrane materials.
  • the method of transmembrane reduces the particle size of the liposome, and separately encloses drugs such as DiR, FAM, and doxorubicin (DOX) to construct liposomes having an average particle diameter of about 100 nm.
  • drugs such as DiR, FAM, and doxorubicin (DOX) to construct liposomes having an average particle diameter of about 100 nm.
  • the particle size distribution was measured by dynamic light scattering method, and the liposome morphology was observed by negative staining electron microscopy.
  • D A7R-PEG-Liposome/DiR, cA7R-PEG-Liposome/DiR, L A7R-PEG-Liposome/DiR and mPEG-Liposome were injected into the tail vein of nude mice bearing the U87 subcutaneous xenograft model. /DiR, comparing the intratumoral distribution of different drug delivery systems at various time points.
  • D A7R-PEG-Liposome/Doxorubicin, cA7R-PEG-Liposome/Doxorubicin, L A7R-PEG-Liposome/Doxorubicin were injected into the tail vein of nude mice bearing the U87 subcutaneous xenograft model.
  • mPEG-liposome/adriamycin, free doxorubicin and normal saline the tumor volume, tumor weight and tumor tissue apoptosis, neovascularization and mimicry of blood vessels as indicators to evaluate the different doxorubicin delivery system Anti-tumor effect in vivo.
  • the U7 subcutaneous xenograft model was injected into the tail vein of nude mice with D A7R, L A7R, D A7R and DOX, L A7R and DOX, DOX and normal saline.
  • the tumor anti-tumor effect was evaluated by tumor volume and tumor weight.
  • the A549 subcutaneous transplantation tumor model was injected with D A7R, D A7R and DOX, D A7R and LS/DOX, DOX, LS/DOX and normal saline in the tail vein of the nude mice.
  • the tumor anti-tumor effect was evaluated by the tumor volume and tumor weight.
  • D A7R and DOX, L A7R and DOX and free doxorubicin were injected into the tail vein of nude mice bearing subcutaneous transplantation tumor model. After 1 hour, the tumors were taken out to make frozen sections, CD31 labeled blood vessels, DAPI stained nuclei, and the drugs in each group were compared. Internal distribution.
  • mice Normal mice were injected with D A7R, D A7R-Aldoxorubicin, D A7R and DOX, Aldoxorubicin, and DOX in the tail vein to investigate the maximum tolerated dose (MTD).
  • MTD maximum tolerated dose
  • test results of the present invention indicate that: D A7R and cA7R have higher stability in serum than L A7R, and have similar affinity to VEGFR2 and NRP-1 receptor proteins, and a model for highly expressing these two receptors.
  • the cell affinity activity is similar, but it has better tumor tissue targeting ability and imaging effect in the model animal; compared with the L A7R modified drug complex or the nano drug delivery system, the D A7R or cA7R modified drug complex or Nano drug delivery systems have shown better tumor targeting performance and stronger anti-tumor effects.
  • the present invention provides cA7R and D A7R with significant antitumor activity, significantly superior to L A7R. And as a synergistic synergist, combined with anti-tumor drugs, exert a stronger anti-tumor effect.
  • Figure 1 shows the ESI-MS spectrum of D A7R
  • Figure 2 shows the ESI-MS spectrum of D A7R-Cys
  • Figure 3 shows the ESI-MS spectrum of L A7R
  • Figure 4 shows the ESI-MS spectrum of L A7R-Cys
  • Figure 5 shows the ESI-MS spectrum of cA7R
  • Figure 6 shows the ESI-MS spectrum of D A7R-Fluorescein
  • Figure 7 shows an ESI-MS spectrum of L A7R-Fluorescein
  • Figure 8 shows the ESI-MS spectrum of cA7R-Fluorescein
  • Figure 9 shows an ESI-MS spectrum of D A7R-Aldoxorubicin
  • Figure 10 shows an ESI-MS spectrum of L A7R-Aldoxorubicin
  • Figure 11 shows the 1 H-NMR spectrum of D A7R-PEG 3400 -DSPE and cA7R-PEG 3400 -DSPE;
  • Figure 12 shows the particle size and electron micrograph of the doxorubicin-loaded liposome.
  • Figure A and Figure B are the particle size and electron micrograph of each doxorubicin liposome
  • Figure 13 shows the serum stability of D A7R and cA7R
  • Figure 14 shows the binding activity of D A7R and cA7R to VEGFR2
  • Figure 15 shows the binding activity of D A7R and cA7R to NRP-1
  • Figure 16 shows the uptake of Fluorescein-labeled polypeptide by glioma cell line U87
  • Figure A and Figure B are the results of laser confocal photographs and flow cytometry after 4 hours of exposure of Fluorescein-labeled D A7R, cA7R and L A7R to U87 cells;
  • Figure 17 shows the uptake of Fluorescein-labeled polypeptide by HUVEC in umbilical vein endothelial cells.
  • Figure A and Figure B are the results of laser confocal photographs and flow cytometry after 4 hours of exposure of Fluorescein-labeled D A7R, cA7R and L A7R to HUVEC cells;
  • Figure 18 shows the uptake of Fluorescein marker polypeptide by the U87 mimetic vascular in vitro model
  • Figure 19 shows the distribution of the subcutaneous xenograft of the Fluorescein-labeled polypeptide
  • Figure A shows the results of in vitro tumor image distribution after injection of Fluorescein-labeled peptide into the tail vein of nude mice bearing U87 subcutaneously transplanted tumor;
  • Figure B shows the results of image distribution of isolated organs;
  • Figure C shows the semi-quantitative results of fluorescence of isolated tumors;
  • Figure D shows the semi-quantitative results of fluorescence of isolated tumors and organs;
  • Figure 20 shows the uptake of A7R-Aldoxorubicin by glioma cell line U87,
  • Figure A and Figure B show the results of laser confocal photographs and flow cytometry after 4 hours of A7R-Aldoxorubicin and U87 cells;
  • Figure 21 shows the uptake of A7R-Aldoxorubicin by HUVEC in umbilical vein endothelial cells.
  • Figure A and Figure B show the results of laser confocal photographs and flow cytometry after 4 hours of A7R-Aldoxorubicin and HUVEC cells;
  • Figure 22 shows the pH stability of D A7R-Aldoxorubicin and cA7R-Aldoxorubicin
  • Figure 23 shows the serum stability of D A7R-Aldoxorubicin and cA7R-Aldoxorubicin
  • Figure 24 shows the subcutaneous intratumoral distribution of A7R-Aldoxorubicin
  • Figure 25 shows the activity curves of A7R-Aldoxorubicin against U87 cells and HUVEC cells in vitro
  • Figure A and Figure B are the activity curves of D A7R-Aldoxorubicin, L A7R-Aldoxorubicin, Aldoxorubicin and DOX anti-U87 cells and HUVEC cells, respectively;
  • Figure 26 shows the A7R-Aldoxorubicin subcutaneous tumor inhibition experiment.
  • Figure A is a graph showing the tumor volume of each group in nude mice as a function of time.
  • Figure B is a graph showing the body weight of each group in nude mice as a function of time.
  • Figure C is a statistical analysis result of weighing the nude mice after removing the tumor tissue.
  • Figure 27 shows U87 subcutaneous tumor inhibition experiments at different doses of A7R-Aldoxorubicin.
  • Figure A is a graph showing the tumor volume of each group in nude mice as a function of time.
  • Figure B is a graph showing the body weight of each group in nude mice as a function of time.
  • Figure C is a statistical analysis result of weighing the nude mice after removing the tumor tissue.
  • Figure 28 shows the results of CD31/PAS staining and TUNEL staining
  • Figure 29 shows the U87 subcutaneous tumor suppressor immunohistochemistry test of different doses of A7R-Aldoxorubicin.
  • Figure 30 shows the systemic toxicity of A7R-Aldoxorubicin
  • Figure 31 shows the uptake of 5-87 M polypeptide-modified liposomes by U87 cells.
  • Figure A and Figure B are the results of laser confocal photographs and flow cytometry after 4 h of D A7R, cA7R and L A7R modified liposomes loaded with fluorescein 5-FAM and U87 cells, respectively.
  • Figure 32 shows uptake of encapsulated 5-FAM liposomes by HUVEC cells
  • Figure A and Figure B are the laser confocal photographs and flow results of the liposomes loaded with 5-FAM at 37 °C for 4 h after treatment with HUVEC cells, respectively;
  • Figure 33 shows the uptake of the entrapped 5-FAM liposomes by the U87 mimetic vascular in vitro model
  • Figure 34 shows the intradermal distribution of PEG-liposome loaded with a near-infrared dye
  • Figure A shows the in-vivo fluorescence distribution image after 24 hours of tail vein injection.
  • Figure B shows the semi-quantitative results of intratumoral fluorescence intensity at various time points after administration.
  • Figure C shows the fluorescence distribution image of the organ, and Figure D shows the fluorescence of Figure C. Semi-quantitative statistical results;
  • Figure 35 shows the activity curves of anti-U87 cells and HUVEC cells in vitro loaded with doxorubicin liposomes
  • Figure A and Figure B are the activity curves of D A7R-LS/DOX, cA7R-LS/DOX, L A7R-LS/DOX, LS/DOX and DOX anti-U87 cells and HUVEC cells, respectively;
  • Figure 36 shows the inhibition of neovascularization by doxorubicin-loaded liposomes in vitro
  • Figure A is a photograph of the inhibition of neovascularization in vitro by D A7R-LS/DOX, cA7R-LS/DOX, L A7R-LS/DOX, LS/DOX, and DOX.
  • Figure B shows the statistical results of the formation rate of vascular-like structures in each group. ;
  • Figure 37 shows the inhibition of mimic angiogenesis by doxorubicin-loaded liposomes in vitro
  • Figure A shows the inhibition of D A7R-LS/DOX, cA7R-LS/DOX, L A7R-LS/DOX, LS/DOX, and DOX on the mimic vascular in vitro model
  • Figure B shows the statistical results of the formation rate of mimetic vascular structures in each group.
  • Figure 38 shows a subcutaneous tumor inhibition test of doxorubicin-loaded liposomes
  • Figure A is a graph showing the tumor volume of each group of nude mice as a function of time
  • Figure B is a statistical analysis result of weighing the nude mice after removing the tumor tissue
  • Figure 40 shows the results of CD31/PAS double staining
  • Figure 41 shows a subcutaneous tumor inhibition test in combination with A7R and A7R and DOX.
  • Figure A is a graph showing the tumor volume of each group in nude mice as a function of time.
  • Figure B is a graph showing the body weight of each group in nude mice as a function of time.
  • Figure C is a statistical analysis result of weighing the nude mice and removing the tumor tissue.
  • Figure 42 shows D A7R and D A7R were combined with DOX to administer an anti-U87 orthotopic tumor pharmacodynamic test;
  • Figure 43 shows the pharmacodynamic test of anti-U87 orthotopic tumor in combination with D A7R and LS/DOX;
  • Figure 44 shows the pharmacodynamic test of the combination of D A7R and D A7R with TMZ
  • Figure 45 shows the pharmacodynamic test of D A7R and D A7R in combination with DOX against A549 subcutaneous tumor
  • Figure 46 shows the subcutaneous intratumoral distribution of U87 in combination with A7R and DOX
  • FIG 47 shows the maximum tolerated dose (MTD) test
  • Figure A is a different doses DOX mice body weight change curve
  • curve B of FIG different doses Aldoxorubicin mice body weight body weight change
  • Figure C D A7R mice curve D in FIG different doses of different doses D
  • the body weight change curve of mice in combination with A7R and DOX Figure E is the body weight change curve of D A7R-Aldoxorubicin mice at different doses
  • Figure F is the bar graph of body weight change of each group on the 8th day.
  • Example 1 Synthesis and characterization of L A7R and D A7R, L A7R-Cys and D A7R-Cys
  • D A7R sequence D R D P D P D L D W D T D A
  • D A7R-Cys sequence D
  • D R D P D P D L D W D T D A design and synthesize D A7R (sequence D R D P D P D L D W D T D A) and D A7R-Cys (sequence D ) composed of non-natural D-configuration amino acids by reverse-phase solid phase peptide synthesis C D R D P D P D P D L D W D T D A).
  • L A7R sequence is ATWLPPR
  • L A7R-Cys sequence is ATWLPPRC
  • PAM-Boc resin was deprotected with trifluoroacetic acid (TFA).
  • TFA trifluoroacetic acid
  • the amino acid was protected by Boc in a sequential manner.
  • the boronic acid was decarboxylated, and the resin was washed with DMF, DCM/MeOH (1:1, v/v) and dried in vacuo.
  • the resin was placed in a peptide cutting tube, an appropriate amount of P-cresol was added, then hydrogen fluoride was introduced, and the reaction was stirred for 1 hour in an ice bath. After the completion of the reaction, the hydrogen fluoride in the tube was removed under reduced pressure, and the precipitate was washed three times with ice diethyl ether.
  • the residual precipitate was dissolved in 20% acetonitrile, and the filtrate was collected and then evaporated to give a crude peptide solution.
  • the crude peptide was isolated and purified by acetonitrile/water (containing 0.1% TFA) system.
  • the linear polypeptide A7R-Mpr-Leu was designed and synthesized by solid peptide synthesis.
  • the target product cA7R was then synthesized using the "Native Chemical Ligation" reaction.
  • the purified A7R-Mpr-Leu was dissolved in 6M guanidine hydrochloride solution at a concentration of 1 mg/mL, and 1 ⁇ (v/v) of catalyst thiophenol was added, and the reaction was stirred and sealed, and the progress of the reaction was confirmed by HPLC until the peak of the raw material disappeared. When the peak height of the product no longer increased, 20% piperidine was added to remove the termination reaction.
  • the D A7R-Cys, L A7R-Cys or cA7R obtained in Example 1 or Example 2 was dissolved in 0.1 M PBS solution (pH 7.2), and Fluorescein-5-maleimide was dissolved in DMF. The reaction was stirred and monitored by HPLC. After the reaction of D A7R-Cys, L A7R-Cys or cA7R was completed, the reaction was stopped, purified by liquid phase, and purified by acetonitrile/water (containing 0.1% TFA) system. Freeze-dried D A7R-Fluorescein, L A7R-Fluorescein or cA7R-Fluorescein pure product.
  • Maleimide-DTPA is dissolved in DMF, and mixed with D A7R-Cys, L A7R-Cys or cA7R in PBS, stirred for reaction, prepared for liquid phase purification, and lyophilized to obtain D A7R-DTPA, L A7R-DTPA or cA7R-DTPA. Pure product, chelated Gd will get A7R-DTPA-Gd.
  • An example of linking a ketone- or aldehyde-containing drug as A7R was prepared using an A7R-doxorubicin conjugate. 9.4 mg of the thiol-containing A7R polypeptide was dissolved in 3 mL of phosphate buffer (0.1 mM, pH 7.0) and stirred at 4 ° C for 20 min. Then, a 4-fold molar amount of doxorubicin 6-maleimido hexanide derivative was added and reacted at room temperature in the dark for 1 h.
  • the reaction solution was separated by a C18 preparative column (column: waters X bridge 19 ⁇ 300 mm; mobile phase: A 0.01 M pH 7.4 phosphate buffer, B acetonitrile; elution method: 100% A to 100% B linear gradient), The corresponding fractions were collected, desalted and lyophilized to obtain D A7R or L A7R-doxorubicin conjugate (i.e., D A7R-Aldoxorubicin or L A7R-Aldoxorubicin).
  • the mass spectrum is shown in Figures 9 and 10, and the ESI-MS is 1692.6, which is consistent with the theoretical molecular weight.
  • A7R-paclitaxel complex is used as a A7R to link a drug with a disulfide bond.
  • the paclitaxel 3-(2-pyridinyl)propionic acid derivative was dissolved in 5 mL of DMF, 1.5-fold molar amount of sulfhydryl-containing A7R was dissolved in PBS/DMF, and the pH of the solution was maintained at 4-5 to treat paclitaxel 3-(2- The pyridinyldipropionate derivative is added dropwise to the thiol-polypeptide solution, reacted at room temperature for 6 hours, and prepared by lyophilization to obtain an A7R-paclitaxel complex.
  • the A7R-bortezomib complex was used as an example of the A7R nitrogen-terminal modified drug.
  • amino acids are sequentially inserted on the resin, and all amino acid residues of the polypeptide are completed, and the boronic acid of the nitrogen end is removed by trifluoroacetic acid.
  • a DMF solution containing 3 times the molar amount of succinic anhydride and DIEA was added and reacted at room temperature for 30 min. After washing the resin, dopeamine was protected by adding 5-fold molar amount of trimethylchlorosilane, and reacted with HBTU/DIEA as a condensing agent for 1 hour at room temperature.
  • the resin was cut with HF and purified by preparative HPLC to obtain a polypeptide-dopamine derivative.
  • the polypeptide-dopamine derivative was mixed with bortezomib at a molar ratio of 1:1 to obtain an A7R-bortezomib complex.
  • the A7R-p53 activating peptide PMI complex was used as an example of an A7R fusion polypeptide drug.
  • the method is directly prepared by a solid phase polypeptide synthesis method. After the A7R-PMI polypeptide sequence is determined, the amino acid is sequentially inserted in the same manner as the preparation of A7R, and the A7R-PMI fusion polypeptide is obtained by cleavage and purification by HF.
  • the nuclear magnetic spectrum of Mal-PEG-DSPE showed a maleimide peak at 6.7 ppm, while the peak disappeared in the nuclear magnetic spectrum of D A7R-PEG-DSPE, L A7R-PEG-DSPE and cA7R-PEG-DSPE, indicating that Mal- The maleimide group in the PEG-DSPE has been attached to A7R.
  • the A7R-PEG-liposome membrane material is formulated as HSPC/Chol/mPEG 2000 -DSPE/A7R-PEG-DSPE (52:43:3:2, mol/mol), and the preparation process for different properties is slightly different.
  • Water-soluble drug The above membrane material was weighed and dissolved in chloroform, and the organic solvent was removed by rotary evaporation under reduced pressure to obtain a uniform lipid film, which was dried under vacuum for 24 h.
  • the aqueous solution of water-soluble fluorescein (5-FAM) or nuclear magnetic imaging agent (Gd-DTPA) was added to hydrate and shaken in a water bath at 60 ° C for 2 hours to obtain a liposome suspension.
  • use a high-pressure homogenizer if the liposome volume is less than 10 mL, use a micro-extruder
  • unencapsulated 5-FAM or Gd-DTPA was separated by using a physiological saline as an eluate on a Sephadex G-50 column to obtain a liposome encapsulating 5-FAM or Gd-DTPA.
  • Hydrophobic drug The membrane material and the hydrophobic near-infrared dye (DiR) were dissolved in chloroform, and the organic solvent was removed by rotary evaporation under reduced pressure to obtain a uniform lipid film, which was dried under vacuum for 24 h.
  • the physiological saline solution was added to hydrate and shaken in a water bath at 60 ° C for 2 hours to obtain a liposome suspension.
  • the post-treatment was the same as above to obtain a liposome containing DiR.
  • doxorubicin liposomes are prepared by ammonium sulfate gradient method. The particle size distribution was measured by dynamic light scattering (Fig. 12A), and the liposome morphology was observed by negative staining electron microscopy (see Fig. 12B). It can be seen from the figure that there is no significant difference in the size and morphology of A7R modified liposomes and unmodified liposomes.
  • D A7R, cA7R and L A7R were formulated into 1 mg/mL aqueous solution, 0.1 mL was added to 0.9 mL of 25% mouse serum, and incubated at 37 ° C, 100 ⁇ L of reaction solution was taken at 0, 15 min, 0.5, 1, 2 and 4 h, respectively.
  • the protein in the serum was precipitated by adding 20 ⁇ L of acetonitrile, allowed to stand at 4 ° C for 20 min, centrifuged at 12,000 rpm for 10 min, and 20 ⁇ L of the supernatant was taken for HPLC analysis. See Figure 13 for serum stability results.
  • the ordinate of the graph is the residual percentage of intact polypeptide.
  • Test Example 2 Experimental study on binding activity of A7R to vascular endothelial growth factor receptor 2 (VEGFR2)
  • Pre-binding analysis was performed by the biacore system, and pH 4.5 was selected as the optimal VEGFR2 protein to bind pH to the CM5 chip.
  • the recombinant human VEGFR2 protein was coupled to a CM5 chip and the RU value reached the target value.
  • D A7R, cA7R, and L A7R were separately disposed as sample solutions at concentrations of 5, 10, 20, 40, 80, and 160 nM. From low to high, the Biacore T200Evaluation software was used to analyze the binding activities of D A7R, cA7R and L A7R to VEGFR2 protein, and their K D values were calculated (Fig. 14). D A7R, cA7R and L A7R were observed. VEGFR2 protein binding activity similar, K D values of 8.414nM, 6.794nM and 9.289nM.
  • Test Example 3 Experimental study on binding activity of A7R to neuropilin-1 (NRP-1)
  • Pre-binding analysis was performed by the biacore system, and pH 4.5 was selected as the optimal NRP-1 protein to bind pH to the CM5 chip.
  • Recombinant human NRP-1 protein was coupled to the CM5 chip and the RU value reached the target value.
  • D A7R, cA7R, and L A7R were each disposed as a sample solution having a concentration of 2.5, 5, 10, 20, 40, and 80 nM.
  • the samples were injected from low to high, and the binding activities of D A7R, cA7R and L A7R with NRP-1 protein were analyzed by Biacore T200Evaluation software software, and their K D values were calculated (Fig. 15).
  • D A7R, cA7R and L were observed.
  • the binding activity of A7R to NRP-1 protein was similar, with K D values of 2.31 nM, 10.57 nM and 6.62 nM, respectively.
  • Test Example 4 In vitro targeting of glioma cell line U87 by A7R
  • Monolayer cultured glioma cells (U87 cells) in logarithmic growth phase were digested with 0.25% trypsin, and mixed with DMEM medium containing 10% fetal bovine serum to prepare a single cell suspension.
  • 1 ⁇ 10 5 cells per well were seeded in a 12-well culture plate at a volume of 1 mL per well.
  • the culture plate was transferred to a carbon dioxide incubator, and cultured at 37 ° C, 5% CO 2 and saturated humidity for 24 hours, with 10%.
  • a DMEM medium of fetal bovine serum was prepared at a concentration of 5 ⁇ M of FAM, D A7R-Fluorescein, cA7R-Fluorescein and L A7R-Fluorescein solutions.
  • the culture solution in the culture plate was aspirated, and the above solution was separately added, and the mixture was incubated at 37 ° C for 4 hours, and the supernatant was aspirated. Wash the cells three times with PBS solution, fix the cells with formaldehyde fixative, and stain the cells with DAPI. After laser confocal observation, the internalization of the cells is shown in Figure 16A. After washing three times with PBS, flow cytometry analysis was carried out, and the results are shown in Fig. 16B. It can be seen that the uptake of D A7R, cA7R and L A7R by U87 cells is significantly higher than that of free fluorescein, but there is no significant difference in the uptake of the three polypeptides.
  • Test Example 5 In vitro targeting of human umbilical vein endothelial cells HUVEC by A7R
  • VEC cells Mononuclear cultured human umbilical vein endothelial cells
  • monolayer cultured cells were digested with 0.25% trypsin, and mixed with DMEM medium containing 10% fetal bovine serum to prepare a single cell suspension.
  • DMEM medium containing 10% fetal bovine serum
  • 1 ⁇ 10 5 cells per well were seeded in a 12-well culture plate at a volume of 1 mL per well. The culture plate was transferred to a carbon dioxide incubator, and cultured at 37 ° C, 5% CO 2 and saturated humidity for 24 hours, with 10%.
  • the DMEM medium of fetal bovine serum was prepared at a concentration of 5 ⁇ M of FITC, D A7R-Fluorescein, cA7R-Fluorescein and L A7R-Fluorescein solutions.
  • the culture solution in the culture plate was aspirated, and the above solution was separately added, and the mixture was incubated at 37 ° C for 4 hours, and the supernatant was aspirated. Wash the cells three times with PBS solution, fix the cells with formaldehyde fixative, and stain the cells with DAPI. After laser confocal observation, see Figure 17A for cell internalization. After washing three times with PBS, flow cytometry analysis was carried out, and the results are shown in Fig. 17B. It can be seen that the uptake of D A7R, cA7R and L A7R by HUVEC cells is significantly higher than that of free fluorescein, but there is no significant difference in the uptake of the three polypeptides.
  • Test Example 6 In vitro targeting of A7R to U87 mimetic vascular model in vitro
  • a solution of FITC, D A7R-Fluorescein, cA7R-Fluorescein and L A7R-Fluorescein at a concentration of 5 ⁇ M was prepared in DMEM medium containing 10% fetal bovine serum.
  • the culture solution in the culture plate was aspirated, and the above solution was separately added, and the mixture was incubated at 37 ° C for 4 hours, and the supernatant was aspirated. Wash the cells three times with PBS solution, fix the cells with formaldehyde fixative, and stain the cells with DAPI. Observe by fluorescence microscope. See photo in Figure 18.
  • Test Example 7 A7R in vivo tumor targeting validation
  • a subcutaneous tumor animal model was constructed. U87 cells in the logarithmic growth phase were trypsinized, adjusted to a cell concentration of 3 ⁇ 10 7 cells/mL, and inoculated with 100 ⁇ L to the right abdomen of the nude mice. After inoculation, the animals were raised in the SPF class, and the tumor size was observed regularly. When the tumor size was 200 mm 3 , the tumor-bearing nude mice without necrosis and regular tumor shape were selected and tested in groups.
  • the FITC, D A7R-Fluorescein, cA7R-Fluorescein and L A7R-Fluorescein solutions were injected into the tumor-bearing nude mouse model through the tail vein at a dose of 0.15 ⁇ mol/mouse. After 1 h, the nude mice were sacrificed and the tumor was removed and detected by a live imager. The fluorescence distribution of the tumor is shown in Figure 19.
  • Figure A shows the results of in vitro tumor image distribution after injection of Fluorescein-labeled peptide into the tail vein of nude mice bearing U87 subcutaneously transplanted tumor;
  • Figure B shows the results of image distribution of isolated organs;
  • Figure C shows the semi-quantitative results of fluorescence of isolated tumors;
  • Panel D is the semi-quantitative fluorescence of isolated tumors and organs.
  • the accumulation of Fluorscein-labeled D A7R, cA7R and L A7R in tumors was significantly higher than that of free fluorescein (*p ⁇ 0.05, **p ⁇ 0.005), and the tumor targeting effects were: D A7R>cA7R> L A7R.
  • Test Example 8 In vitro targeting of glioma cell line U87 by A7R-Aldoxorubicin
  • Monolayer cultured glioma cells (U87 cells) in logarithmic growth phase were digested with 0.25% trypsin, and mixed with DMEM medium containing 10% fetal bovine serum to prepare a single cell suspension.
  • 1 ⁇ 10 5 cells per well were seeded in a 12-well culture plate at a volume of 1 mL per well.
  • the culture plate was transferred to a carbon dioxide incubator, and cultured at 37 ° C, 5% CO 2 and saturated humidity for 24 hours, with 10%.
  • a DMEM medium of fetal bovine serum was prepared at a concentration of 5 ⁇ M of DOX, Aldoxorubicin, D A7R-Aldoxorubicin and L A7R-Aldoxorubicin solutions.
  • the culture solution in the culture plate was aspirated, and the above solution was separately added, and the mixture was incubated at 37 ° C for 4 hours, and the supernatant was aspirated. Wash the cells three times with PBS solution, fix the cells with formaldehyde fixative, and stain the cells with DAPI. After laser confocal observation, the photos of cell internalization are shown in Fig. 20A. After washing three times with PBS, flow cytometry analysis was carried out, and the results are shown in Fig. 20B. It can be seen that U87 cells have uptake of A7R-Aldoxorubicin.
  • Test Example 9 In vitro targeting of human umbilical vein endothelial cells HUVEC by A7R-Aldoxorubicin
  • HUVEC cells Human umbilical vein endothelial cells (HUVEC cells) cultured in monolayers in logarithmic growth phase were tested as above, and the internalization photographs are shown in Fig. 21A. The flow cytometry results are shown in Figure 21B. It can be seen that HUVEC cells have uptake of A7R-Aldoxorubicin.
  • Test Example 10 Stability of A7R-Aldoxorubicin at different pHs
  • D A7R-Aldoxorubicin and L A7R-Aldoxorubicin were separately formulated into 1 mg/mL phosphate solution (pH 5.5, 6.5, 7.4) and incubated at 37 ° C for 0, 0.5, 1, 2, 4, 8, 12 and 24 h respectively. 20 ⁇ L of the solution was taken out and subjected to HPLC analysis.
  • the pH stability results indicate that L A7R-Aldoxorubicin (Fig. 22A) and D A7R-Aldoxorubicin (Fig. 22B) are most stable at pH 7.4, with the fastest hydrolysis at pH 5.5 and weakly acidic conditions (pH 6.5). It also degrades well, indicating that the A7R-Aldoxorubicin linked by hydrazone can release free doxorubicin in a weakly acidic environment.
  • Test Example 11 Stability of A7R-Aldoxorubicin in serum
  • D A7R-Aldoxorubicin and L A7R-Aldoxorubicin were formulated into 1 mg/mL aqueous solution, 0.1 mL was added to 0.9 mL of 25% mouse serum, and incubated at 37 ° C at 0, 0.25, 0.5, 1, 2, 4, and 8, respectively. 100 ⁇ L of the reaction solution was taken out at 12 h, 20 ⁇ L of acetonitrile was added to precipitate the serum protein, and the mixture was allowed to stand at 4 ° C for 20 min, centrifuged at 12,000 rpm for 10 min, and 20 ⁇ L of the supernatant was taken for HPLC analysis. As can be seen from the figure (Fig.
  • Test Example 12 Tumor targeting validation of A7R-Aldoxorubicin in vivo
  • D A7R-Aldoxorubicin, L A7R-Aldoxorubicin, Aldoxorubicin and DOX were injected into the tumor-bearing nude mouse model at a dose of 10 mg/Kg. After 1 h, the nude mice were sacrificed and the tumor was removed. The OCT embedding agent (Tissue-Tek) was used. Embedding, rapid freezing in liquid nitrogen, making 10 ⁇ m frozen sections, fixed in acetone at 4 ° C for 10 min, washed with PBS, and incubated with bovine serum albumin (BSA) for 1 h.
  • BSA bovine serum albumin
  • Sections were incubated with rat anti-mouse CD31 (1:10) for 1 h, then incubated with FITC-labeled goat anti-rat IgG (1:100) to localize tumor blood vessels, and finally sections were counterstained with DAPI to reveal nuclei. After sealing, it was observed with a laser confocal microscope (Fig. 24). The results showed that A7R-Aldoxorubicin accumulated better in tumor tissues than Aldoxorubicin and DOX, and could co-localize with neovascularization. The targeting effect of D A7R-Aldoxorubicin is better than that of L A7R-Aldoxorubicin.
  • Test Example 13 In vitro efficacy test of A7R-Aldoxorubicin
  • Panels A and B are the activity curves of D A7R-Aldoxorubicin, L A7R-Aldoxorubicin, Aldoxorubicin and DOX anti-U87 cells and HUVEC cells, respectively.
  • Panel A shows that administration of U87 cells were cultured 72h, the 6.76,13.18,2.95 an IC 50, respectively, and 0.081 ⁇ M. All four compounds inhibited the growth of U87 cells in vitro.
  • Figure B shows that administration of HUVEC cell culture 72h, an IC 50 and respectively 0.63,1.047,0.891 0.0105 ⁇ M. All four compounds inhibited the growth of HUVEC cells in vitro.
  • Test Example 14 Inhibition test of subcutaneous xenografts by A7R-Aldoxorubicin
  • U87 subcutaneous tumor animal model was constructed, and the tumor size was observed regularly.
  • the tumor size was 100 mm 3
  • the test was performed in groups, and D A7R-Aldoxorubicin, L A7R-Aldoxorubicin, Aldoxorubicin, DOX and physiological saline were injected into the tail vein respectively.
  • the total dose of doxorubicin in the administration group was 2.5 mg/kg, which was divided into five times, and the interval between each administration was two days.
  • the long diameter (a) and short diameter (b) of the tumor were measured by vernier calipers the next day.
  • the tumor volume of each group of nude mice was calculated according to the formula, and the curve of tumor volume with time was plotted. The statistical differences of each group were calculated. Calculate the tumor volume according to the following formula:
  • V tumor volume 0.5 (a ⁇ b 2)
  • Figure A is a graph showing the tumor volume of each group in nude mice as a function of time.
  • Figure B is a graph showing the body weight of each group in nude mice as a function of time.
  • Figure C is a statistical analysis of the weight of the nude mice after the tumors were removed and weighed. The antitumor effect of A7R-Aldoxorubicin is better than that of unmodified DOX, and D A7R-Aldoxorubicin is the most effective.
  • Test Example 15 Inhibition of subcutaneous xenografts by different doses of A7R-Aldoxorubicin
  • FIG. 27 The high, medium and low doses of the drug-administered group were 2.5 mg/kg, 7.5 mg/kg, 22.5 mg/kg according to the total dose of doxorubicin, and the dose of polypeptide D A7R was 12 mg/kg, the same experiment.
  • Figure A is a graph showing the tumor volume of each group in nude mice as a function of time.
  • Figure B is a graph showing the body weight of each group in nude mice as a function of time.
  • Figure C is a statistical analysis of the weight of the nude mice after the tumors were removed and weighed.
  • the anti-tumor effect of D A7R-Aldoxorubicin was better than DOX at doses of 2.5 mg/kg and 7.5 mg/kg.
  • Test Example 16 A7R-Aldoxorubicin inhibits tumor vasculature and promotes apoptosis
  • D A7R-Aldoxorubicin, L A7R-Aldoxorubicin, Aldoxorubicin, and DOX groups were sacrificed after five times of tail vein injection, and subcutaneous tumors were removed and embedded in paraffin.
  • the angiogenesis inhibitory effect was examined by CD31 immunohistochemical staining and PAS double staining.
  • the degree of apoptosis of tumor cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) using terminal deoxynucleotidyl transferase (TDT).
  • paraffin section is routinely dewaxed to water; PBS is rinsed 3 times for 3 min each time; 0.3% H 2 O 2 solution is treated at room temperature for 20 min; 20 ⁇ g/mL proteinase K is digested for 20 min at 37 ° C; PBS rinses 3 times for 3 min each time 30 ⁇ L of TUNEL mixture (TDT and biotin-dNTP) was added to each section and placed in a humid box for 60 min at 37 ° C; PBS rinsed for 3 min for 3 times; Streptavidin-HRP (1:200) for 30 min at 37 ° C; PBS rinse 3 Times, each time 3 min; 0.04% DAB + 0.03% H 2 O 2 solution developed color for 10 min, washed with water; hematoxylin lining for 1 min, washed with blue; after drying, the conventional resin was sealed.
  • TUNEL mixture TUNEL mixture
  • FIG. 28 The results are shown in Figure 28.
  • D A7R-Aldoxorubicin inhibited the formation of new blood vessels more significantly.
  • D A7R-Aldoxorubicin promoted apoptosis of tumor tissues more significantly.
  • Test Example 17 Different doses of A7R-Aldoxorubicin inhibit tumor vasculature and promote apoptosis
  • the positive nuclei of apoptosis are brownish yellow or tan.
  • D A7R-Aldoxorubicin promoted apoptosis of tumor tissues more significantly than DOX.
  • Test Example 18 Investigation of systemic toxicity of A7R-Aldoxorubicin
  • the heart, liver, spleen, lung and kidney tissues of each group of mice in the subcutaneous tumor pharmacodynamic test were dissected and fixed in 4% paraformaldehyde in PBS solution, embedded in paraffin, and subjected to HE staining under microscope. Observe and take pictures (Fig. 30). The picture shows the HE staining results of heart, liver, spleen, lung and kidney of nude mice in each group of subcutaneous tumors. It shows that the drugs in each group have no obvious toxicity to the organs of nude mice.
  • Test Example 19 In vitro cell targeting validation of A7R-PEG-liposome
  • Monolayer cultured glioma cells (U87 cells) in logarithmic growth phase were digested with 0.25% trypsin, and mixed with DMEM medium containing 10% fetal bovine serum to prepare a single cell suspension.
  • 1 ⁇ 10 5 cells per well were seeded in a 12-well culture plate at a volume of 1 mL per well. The culture plate was transferred to a carbon dioxide incubator, and cultured at 37 ° C, 5% CO 2 and saturated humidity for 24 hours, with 10%.
  • DMEM medium of fetal bovine serum was prepared at a concentration of 5 ⁇ M of mPEG-liposome/FAM, D A7R-PEG-liposome/FAM, cA7R-PEG-liposome/FAM and L A7R-PEG-liposome/ FAM solution.
  • the culture solution in the culture plate was aspirated, and the above solution was separately added, and the mixture was incubated at 37 ° C for 4 hours, and the supernatant was aspirated. Wash the cells three times with PBS solution, fix the cells with formaldehyde fixative, and stain the cells with DAPI. After laser confocal observation, the photos of cell internalization are shown in Figure 31A.
  • Test Example 20 In vitro targeting of human umbilical vein endothelial cells HUVEC by A7R-PEG-liposome
  • Fig. 32A Single-layer cultured human umbilical vein endothelial cells (HUVEC cells) in the logarithmic growth phase were tested as above, and the internalization photographs are shown in Fig. 32A, and the flow cytometry results are shown in Fig. 32B. As can be seen from the figure, the uptake of polypeptide-modified liposomes by HUVEC cells was significantly higher than that of polypeptide-free modified liposomes.
  • Test Example 21 In vitro targeting of U7 mimetic vascular model of U87 in vitro by A7R-PEG-liposome
  • mPEG-Liposome/FAM, D A7R-PEG-Liposome/FAM, cA7R-PEG-Liposome/FAM and L A7R-PEG- at a concentration of 5 ⁇ M were prepared in DMEM medium containing 10% fetal bovine serum. Liposomal/FAM solution. The culture solution in the culture plate was aspirated, and the above solution was separately added, and the mixture was incubated at 37 ° C for 4 hours, and the supernatant was aspirated. Wash the cells three times with PBS solution, fix the cells with formaldehyde fixative, and stain the cells with DAPI. Observe by fluorescence microscope. See photo in Figure 33 for photos. As can be seen from the figure, the U87 mimetic vascular in vitro model is significantly higher than the polypeptide-free modified liposome.
  • Test Example 22 In vivo targeting verification of A7R-PEG-liposome
  • U87 subcutaneous tumor model nude mice were injected with 100 ⁇ L of DiR-loaded liposomes, mPEG-liposome/DiR, D A7R-PEG-liposome/DiR, cA7R-PEG-liposome/DiR and L A7R, respectively.
  • PEG-liposome/DiR solution was anesthetized at 2, 4, 8, 12 and 24 h after injection, and the distribution of DiR fluorescence in nude mice was recorded by in vivo imager and semi-quantitative calculation of fluorescence was performed. 34).
  • Panel A is an in vivo fluorescence distribution image after 24 hours of tail vein injection.
  • Panel B is a semi-quantitative result of intratumoral fluorescence intensity at various time points after administration.
  • Figure C is a fluorescence distribution image of an organ.
  • Figure D is a semi-quantitative statistical result of fluorescence of Figure C. The results indicate that D A7R or cA7R modified liposomes are better targeted to the tumor site.
  • Test Example 23 In vitro pharmacodynamic test of A7R-PEG-liposome loaded with doxorubicin
  • U87 cells or HUVEC cells were seeded in 96-well plates at 4.0 ⁇ 10 3 cells/well. After 24 hours, the culture solution was aspirated, and 200 ⁇ L of a series of concentrations of mPEG-liposome/DOX, D A7R-PEG-liposome were added.
  • Panels A and B are the activity curves of LS/DOX, D A7R-LS/DOX, cA7R-LS/DOX, L A7R-LS/DOX and DOX anti-U87 cells and HUVEC cells, respectively.
  • Panel A shows that administration of the U87 cell culture 4h after 72h, an IC 50, respectively 17.78,0.62,0.81,5.13 and 0.06 ⁇ M. All of the four liposomes inhibited the growth of U87 cells in vitro, and the in vitro activities of D A7R-LS/DOX and cA7R-LS/DOX were 8.27 and 6.33 times that of L A7R-LS/DOX, respectively.
  • Panel B shows that the IC 50 of HUVEC cells after 72 h of culture for 4 h was 0.71, 0.19, 0.15, 0.39 and 0.09 ⁇ M, respectively. All of the four liposomes inhibited the growth of HUVEC cells in vitro, and the in vitro activities of D A7R-LS/DOX and cA7R-LS/DOX were 2.05 and 2.60 times, respectively, of L A7R-LS/DOX.
  • Test Example 24 Inhibition test of neonatal blood vessel formation by doxorubicin-containing A7R-PEG-liposome
  • Test Example 25 Inhibition test of mimetic angiogenesis by doxorubicin-containing A7R-PEG-liposome
  • Test Example 26 Inhibition test of subcutaneous xenografts by doxorubicin-loaded A7R-PEG-liposome
  • the U87 subcutaneous tumor animal model was constructed and the tumor size was observed regularly.
  • the tumor size was 100 mm 3
  • the mice were tested in groups, and the tail vein was injected with mPEG-liposome/DOX, D A7R-PEG-liposome/DOX, cA7R-PEG. - liposome/DOX, L A7R-PEG-liposome/DOX, free DOX and physiological saline.
  • the total dose of doxorubicin in the administration group was 10 mg/kg, which was divided into five times, and the interval between each administration was two days.
  • the long diameter (a) and short diameter (b) of the tumor were measured by vernier calipers the next day.
  • the tumor volume of each group of nude mice was calculated according to the formula, and the curve of tumor volume with time was plotted, and the statistical difference of each group was calculated. Calculate the tumor volume according to the following formula:
  • V tumor volume 0.5 (a ⁇ b 2 )
  • Test Example 27 A7R-PEG-liposome apoptosis-promoting assay containing doxorubicin
  • TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling
  • paraffin section is routinely dewaxed to water; PBS is rinsed 3 times for 3 min each time; 0.3% H 2 O 2 solution is treated at room temperature for 20 min; 20 ⁇ g/mL proteinase K is digested for 20 min at 37 ° C; PBS rinses 3 times for 3 min each time 30 ⁇ L of TUNEL mixture (TDT and biotin-dNTP) was added to each section and placed in a humid box for 60 min at 37 ° C; PBS rinsed for 3 min for 3 times; Streptavidin-HRP (1:200) for 30 min at 37 ° C; PBS rinse 3 Times, each time 3 min; 0.04% DAB + 0.03% H 2 O 2 solution developed color for 10 min, washed with water; hematoxylin lining for 1 min, washed with blue; after drying, the conventional resin was sealed.
  • TUNEL mixture TUNEL mixture
  • a positive result is a brown or brown nucleus. Positive brown cells in the nucleus are judged to be apoptotic cells. The number of positive cells in five high power field counts was continuously observed under a common light microscope, and the percentage of positive cells in the cells in the visual field was the apoptotic index. The results are shown in Figure 39.
  • Figure B shows the statistical results of the number of positive cells. Compared with L A7R-LS/DOX, D A7R-LS/DOX and cA7R-LS/DOX can significantly promote the apoptosis of tumor tissues.
  • Test Example 28 Adriamycin-containing A7R-PEG-liposome for tumor angiogenesis test
  • Figure B is the statistical result of the number of new blood vessels. Compared with L A7R-LS/DOX, D A7R-LS/DOX and cA7R-LS/DOX can significantly inhibit the formation of new blood vessels.
  • Test Example 29 Administration of anti-U87 subcutaneous tumor with D A7R polypeptide and D A7R polypeptide in combination with doxorubicin test
  • U87 subcutaneous tumor animal model was constructed, and the tumor size was observed regularly.
  • the tumor size was 100 mm 3
  • the test was performed in groups, and D A7R, L A7R, D A7R and DOX, L A7R and DOX, DOX and physiological saline were injected into the tail vein respectively.
  • the total dose of doxorubicin in the drug-administered group was 2.5 mg/kg, and the total dose of the polypeptide was 20 mg/kg, which was divided into five times, and the interval between each administration was two days.
  • the long diameter (a) and short diameter (b) of the tumor were measured by vernier calipers the next day.
  • the tumor volume of each group of nude mice was calculated according to the formula, and the curve of tumor volume with time was plotted, and the statistical difference of each group was calculated. Calculate the tumor volume according to the following formula:
  • V tumor volume 0.5 (a ⁇ b 2 )
  • Figure A is a graph showing the tumor volume of each group in nude mice as a function of time.
  • Figure B is a graph showing the body weight of each group in nude mice as a function of time.
  • Figure C is a statistical analysis of the weight of the nude mice after the tumors were removed and weighed.
  • the A7R polypeptide itself has antitumor activity, and the D A7R activity is significantly superior to L A7R.
  • Test Example 30 Administration of anti-U87 orthotopic tumors by D A7R polypeptide and D A7R polypeptide in combination with doxorubicin test
  • the U87 in situ tumor model was constructed.
  • the results showed that the median survival of the control group was 20.5 days.
  • the D A7R group was 23.5 days, indicating that the D A7R polypeptide alone can prolong the median survival.
  • the median survival of the DOX group and the D A7R-Aldoxo group were 22.5 days and 25.5 days, respectively, and had a certain therapeutic effect.
  • the median survival of the D A7R and DOX co-administered group was extended to 27 days, reflecting the advantages of the combination.
  • Test Example 31 Combination of D A7R polypeptide and doxorubicin liposome for anti-U87 orthotopic tumor pharmacodynamic test
  • the U87 in situ tumor model was constructed.
  • the results showed that the median survival of the control group was 20.5 days.
  • the D A7R group was 23.5 days, indicating that the D A7R polypeptide alone can prolong the median survival.
  • the median survival time of the LS/DOX group was 26 days, which had a certain therapeutic effect.
  • the median survival of the D A7R and LS/DOX co-administered groups was extended to 31.5 days, reflecting the advantages of combination therapy.
  • Test Example 32 Pharmacodynamic test of D A7R in combination with temozolomide (TMZ)
  • the U87 in situ tumor model was constructed.
  • the sub-dose 4 mg/kg or gavage TMZ single dose 10 mg/kg was used to record the survival time of the model nude mice.
  • the results are shown in Figure 44.
  • the results showed that the median survival of the control group was 25 days.
  • the D A7R group was 26 days, indicating that the D A7R polypeptide alone can prolong the median survival.
  • the median survival of the TMZ group was 43 days, showing a good therapeutic effect.
  • the median survival of the D A7R and TMZ co-administered group was extended to 50 days, reflecting the advantages of the combination.
  • Test Example 33 Pharmacodynamic test of D A7R polypeptide in combination with doxorubicin in anti-A549 subcutaneous tumor
  • the tail vein was injected with D A7R (total dose 20 mg/kg), free doxorubicin (DOX, total dose 5 mg/kg), doxorubicin.
  • DOX free doxorubicin
  • doxorubicin free doxorubicin
  • Liposome LS/DOX, total dose 5 mg/kg
  • D A7R was administered in combination with DOX
  • D A7R was administered in combination with LS/DOX
  • tumor volume of nude mice was monitored every other day, and nude mice were sacrificed on the last day. The tumor is heavy.
  • V tumor volume 0.5 (a ⁇ b 2 )
  • Test Example 34 In vivo tumor targeting validation in combination with A7R and DOX
  • D A7R was administered in combination with DOX, L A7R and DOX in combination with DOX at a dose of 10 mg/kg of doxorubicin, and DOX was injected into the tumor-bearing nude mouse model through the tail vein.
  • the nude mice were sacrificed 1 h later. Tumors were removed, embedded in OCT embedding agent (Tissue-Tek), frozen in liquid nitrogen, and frozen sections of 10 ⁇ m were prepared and fixed in acetone at 4 ° C for 10 min, washed with PBS, and incubated with bovine serum albumin (BSA) for 1 h. .
  • BSA bovine serum albumin
  • Sections were incubated with rat anti-mouse CD31 (1:10) for 1 h, then incubated with FITC-labeled goat anti-rat IgG (1:100) to localize tumor blood vessels, and finally sections were counterstained with DAPI to reveal nuclei. After sealing, it was observed with a laser confocal microscope (Fig. 46).
  • the results showed that compared with L A7R combined with DOX or DOX, D A7R combined with DOX can better accumulate DOX in tumor tissues and colocalize with neovascularization. It is indicated that the combination of D A7R and DOX can increase the accumulation of DOX in the subcutaneous tumor.
  • Test Example 35 Maximum Tolerated Dose (MTD) Test
  • Kunming male mice a group of 3, were administered DOX (5mg/kg; 10mg/kg; 15mg/kg); Aldoxorubicin (5mg/kg according to DOX; 10mg/kg; 15mg/kg);
  • D A7R-Aldoxorubicin (5 mg/kg according to DOX; 10 mg/kg; 15 mg/kg); polypeptide D A7R (10 mg/kg; 20 mg/kg; 30 mg/kg; 40 mg/kg; 50 mg/kg; 60 mg/kg); D A7R DOX administered in combination with (D A7R 8.67mg / kg + DOX 5mg / kg; D A7R 17.35mg / kg + DOX10mg / kg; D A7R 26.02mg / kg + DOX 15mg / kg). Changes in mouse body weight were recorded daily for 8 consecutive days to observe the presence or absence of mouse death. MTD is the dose at which the mice do not die and lose weight ⁇ 15%.
  • Relative weight change daily weight / initial weight ⁇ 100%
  • Figure A is a different doses DOX mice body weight change curve
  • curve B of FIG different doses Aldoxorubicin mice body weight body weight change
  • Figure C D A7R mice curve D in FIG different doses of different doses D
  • the body weight change curve of mice in combination with A7R and DOX Figure E is the body weight change curve of D A7R-Aldoxorubicin mice at different doses
  • Figure F is the bar graph of body weight change of each group on the 8th day.
  • the MTD of the polypeptide D A7R is greater than 60 mg/kg
  • the MTD of DOX is about 10 mg/kg
  • the MTD of D A7R-Aldoxorubicin is about 15 mg/kg.
  • the invention also provides a kit comprising a stabilized A7R polypeptide provided by the invention, a stabilized A7R polypeptide complex or a delivery system.
  • instructions for use may also be included in the kit.
  • the invention further relates to a pharmaceutical composition for diagnosing, tracing and/or treating a tumor comprising a pharmaceutically acceptable carrier, and a stabilized A7R polypeptide provided by the invention, a stabilized A7R polypeptide complex or a drug delivery system.
  • a pharmaceutical composition for diagnosing, tracing and/or treating a tumor comprising a pharmaceutically acceptable carrier, and a stabilized A7R polypeptide provided by the invention, a stabilized A7R polypeptide complex or a drug delivery system.
  • the provided stabilized A7R polypeptide, stabilized A7R polypeptide complex or delivery system can be in an effective amount or in a therapeutically effective amount in the pharmaceutical composition.
  • an effective amount refers to an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals.
  • a “pharmaceutically acceptable” ingredient is suitable for use in humans and/or animals (eg, mammals or birds) without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, has reasonable benefits/ The substance of the risk ratio.
  • “Pharmaceutically acceptable carrier” means a carrier for administration, and may include various excipients, diluents and the like. Such carriers may include, but are not limited to, water, physiological saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, buffers, dextrose, glycerol, ethanol, and Its combination. The choice of carrier should generally be matched to the mode of administration, as is well known to those of ordinary skill in the art.
  • the effective amount of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like.
  • the preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials).
  • the factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, The route of medicine, etc.
  • the invention also provides a method of diagnosing/trace tumors comprising stabilizing an A7R polypeptide complex or delivery system using a stabilized A7R polypeptide provided herein.
  • the invention also provides a method of treating a tumor comprising administering to a subject a therapeutically effective amount of a stabilized A7R polypeptide provided herein, a Stabilizing A7R polypeptide complex or a delivery system.
  • the oral or parenteral administration can be through the gastrointestinal tract, nasal cavity, trachea, lung, non-lesional vein or epidermis, intradermal, subcutaneous, intracardiac, intramuscular, bone marrow, abdominal cavity, epidural, oral, sublingual, and ocular.
  • Preferred modes of administration or modes of administration include oral, respiratory, injection, transdermal, mucosal, or intraluminal administration.
  • the oral administration means includes swallowing, incorporation, and the like.
  • the method of administration of the respiratory tract includes an inhalation method such as ultrasonic atomization inhalation, oxygen atomization inhalation, hand pressure atomization inhalation, and the like.
  • the administration mode of injection includes arterial injection, intravenous injection, intramuscular injection, intracardiac injection, intradermal injection, and the like.
  • the transdermal or transdermal administration methods include iontophoresis, electroporation, and the like.
  • the mucosal administration forms include nasal mucosa administration, oral mucosal administration, ocular mucosal administration, rectal mucosal administration, uterine administration, and vaginal mucosal administration.
  • the method of administration of the lumen includes rectal administration, vaginal administration, urethral administration, nasal administration, ear canal administration, and the like.

Abstract

提供了一种与血管内皮生长因子受体2和神经纤毛蛋白-1高结合活性的逆序D构型多肽DA7R和首尾酰胺键环合多肽cA7R，所述多肽可抗肿瘤。还提供了所述多肽与荧光素、药物、高分子载体材料复合物及其制备方法，以及在肿瘤影像和靶向治疗用递药系统构建中的应用。

Description

稳定化A7R多肽及其在构建肿瘤靶向诊治递药系统中的用途
相关申请的交叉引用
本申请要求2015年10月12日提交的第CN20150653735.9号中国发明专利申请和2016年03月16日提交的第CN201610150524.8号中国发明专利申请的优先权，所述申请以引用的方式整体并入本文。
技术领域
本发明属药学领域，涉及高度稳定且可同时靶向血管内皮生长因子受体2和神经纤毛蛋白-1高表达细胞的逆序D构型多肽和首尾酰胺键环合多肽，其药物复合物和修饰的纳米递药系统以及与抗肿瘤药物联合用药，具体涉及D构型多肽DA7R(D构型氨基酸序列DRDPDPDLDWDTDA)、酰胺键环合多肽cA7R(L构型氨基酸序列c(CATWLPPR))，其诊断和治疗药物复合物、修饰的高分子载体材料所构建的脂质体、聚合物胶束等纳米递药系统，以及在肿瘤诊断和肿瘤靶向治疗中的应用；DA7R、cA7R协同增效抗肿瘤药物的应用。
背景技术
肿瘤是严重威胁人类生命和健康的疾病，死亡率高居所有疾病死亡率首位。传统的化疗作为肿瘤药物治疗的主要手段，存在对肿瘤组织选择性差、毒性大、治疗窗窄、易产生多药耐药等缺陷。为此，为克服传统治疗手段的局限性，近年来纳米递药系统得到越来越多的关注。纳米递药系统具有载药量高、体内循环时间长等优势，其可利用肿瘤的EPR效应，能使药物被动地富集于肿瘤部位，但效率较低。
近年来，主动靶向成为提高肿瘤组织靶向效率的重要策略。主动靶向策略主要针对肿瘤组织中高表达的受体或转运体，利用与特异性受体或转运体具有识别、结合能力的对应配体，将纳米递药系统递送至肿瘤组织或细胞中。常用的对应配体包括单克隆抗体、多肽、核酸适体、小分子化合物等。配体修饰后的纳米递药系统可通过EPR效应富集于肿瘤部位，再通过细胞表面受体或转运体与配体的特异性识别、结合、内化，将药物递送至肿瘤组织和细胞内，从而实现纳米递药系统对肿瘤的主动靶向目标。
血管内皮生长因子受体2(VEGFR2)是血管内皮生长因子(VEGF)的特异性受体，主要高表达于血管内皮细胞和绝大部分肿瘤细胞。
VEGF/VEGFR2信号通路是生理性及病理性血管生成最重要的限速步骤，对肿瘤的血管生成至关重要。VEGF及VEGFR2不仅是目前临床中各种抗肿瘤血管靶向治疗最主要的靶位，而且也是肿瘤学基础研究领域中的热点。最近有研究发现，VEGFR2在脑胶质瘤细胞形成VM过程中起到了关键的作用，提示VEGFR2极有可能成为抗血管新生和拟态血管的共同靶点。神经纤毛蛋白-1(Neuropilin-1，NRP-1)是一种跨膜糖蛋白，是Sema3A和VEGF165的共同受体，在肿瘤新生血管生成、肿瘤生长及转移中发挥重要作用。有研究表明，NRP-1不仅表达于肿瘤血管内皮细胞，同时在多种肿瘤细胞膜上过度表达，包括神经胶质瘤、肺癌、胰腺癌、前列腺癌、乳腺癌和黑色素瘤等。 另外，脑胶质瘤血管的机能失调和高间隙压力，限制了药物穿透血管内皮细胞进入肿瘤实质组织，导致药物的疗效降低。研究发现CendR多肽(由R/KXXR/K氨基酸序列构成的一系列多肽)与NRP-1受体结合后能够增加血管和肿瘤组织通透性，提高了药物或治疗基因穿透进入肿瘤深层组织的能力。
噬菌体展示技术是筛选靶向肿瘤组织有效配体的重要手段之一，通过该技术筛选出来的多肽已经可应用于肿瘤诊断和治疗。LA7R(L构型氨基酸序列ATWLPPR)是通过噬菌体展示技术筛选出的与VEGFR2和NRP-1有高度结合活性的七肽，能靶向VEGFR2和NRP-1高表达的肿瘤新生血管、拟态血管和肿瘤细胞；但LA7R在体内的稳定性较差，在血液中易降解，从而降低了其肿瘤靶向能力。
发明内容
因此，为克服现有技术的缺陷，本发明提供了一种稳定化A7R多肽及其在肿瘤靶向诊治和发挥协同增效抗肿瘤药物的用途，具体涉及制备具有高稳定性的A7R的逆序D构型多肽DA7R(D构型氨基酸序列DRDPDPDLDWDTDA)和首尾酰胺键环合多肽cA7R(L构型氨基酸序列c(CATWLPPR))，并用其修饰药物分子和高分子载体材料，构建稳定性A7R药物复合物、包载药物的稳定性A7R-纳米递药系统，以提高药物对肿瘤的靶向诊疗效果；稳定性A7R本身具有抗肿瘤作用，进一步与抗肿瘤药物联用，具有协同增效的效果。
为了达到上述技术效果，本发明采用了如下的技术方案：
一方面。本发明提供了一种稳定化A7R多肽，所述A7R多肽为逆序D构型多肽DA7R和/或首尾酰胺键环合多肽cA7R，其中，所述逆序D构型多肽DA7R的氨基酸序列为DRDPDPDLDWDTDA，所述首尾酰胺键环合多肽cA7R的L构型氨基酸序列为c(CATWLPPR)。其中，cA7R的氨基酸序列如SEQ ID NO:1所示。具体的，本发明根据多肽逆序合成技术，设计并制备了逆序D构型多肽DA7R，根据“Native Chemical Ligation”反应设计并制备了首尾酰胺键环合多肽cA7R，两条多肽均对血清具有高稳定性、与血管内皮生长因子受体2(VEGFR2)和神经纤毛蛋白-1(NRP-1)具有高亲和力。
另一方面，本发明还提供了一种稳定化A7R多肽复合物，所述稳定化A7R多肽复合物为前述的稳定化A7R多肽修饰含有马来酰亚胺基团的影像物质，其中，所述稳定化A7R多肽复合物的结构为DA7R-X和/或cA7R-X，X为所述影像物质。
优选地，所述X选自荧光物质、近红外染料和磁共振影像剂中的一种或多种，更优选地，所述荧光物质为5-羧基荧光素，所述近红外染料选自cy5.5、IR820和DiR中的一种或多种，所述磁共振影像剂为Gd-DTPA。
具体地，本发明所设计的cA7R和连接半胱氨酸后的DA7R，可利用其分子中巯基与马来酰亚胺功能化荧光物质(FAM)、近红外染料(Cy5.5、IR820、DiR等)和磁共振影像剂(Gd-DTPA，)反应而形成复合物。
又一方面，本发明提供了一种前述稳定化A7R多肽复合物的制备方法，所述方法包括前述的稳定化A7R多肽或巯基化的前述稳定化A7R多肽与含 有马来酰亚胺基团的影像物质反应。
再一方面，本发明提供了一种稳定化A7R多肽复合物，所述稳定化A7R多肽复合物为前述的稳定化A7R多肽修饰抗肿瘤药物，其中，所述稳定化A7R多肽复合物的结构为DA7R-Y和/或cA7R-Y，Y为所述抗肿瘤药物。
优选地，所述抗肿瘤药物选自含酮或醛基的药物、含羟基或氨基的药物、含硼酸基团的药物和多肽药物中的一种或多种。
更优选地：所述含酮或醛基的药物为阿霉素或表阿霉素，所述含羟基或氨基的药物选自紫杉醇、多烯紫杉醇、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、依托泊苷、吉西他滨、阿糖胞苷、5-氟尿嘧啶、替尼泊苷、莫立替尼、埃博霉素、长春瑞滨、放线菌素D、米托蒽醌、丝裂霉素、博来霉素、伊立替康，所述含硼酸基团的药物为硼替佐米或卡非佐米，和/或所述多肽药物选自p53激活肽、蜂毒肽和蝎毒肽中的一种或多种。
具体地，本发明所设计的cA7R和DA7R修饰药物，包括通过马来酰亚胺己肼衍生物反应形成pH敏感腙键(涉及阿霉素、表阿霉素等含酮或醛基的药物)、或通过3-(2-吡啶二巯基)丙酸衍生物反应形成二硫键(涉及紫杉醇、多烯紫杉醇、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、依托泊苷、吉西他滨、阿糖胞苷、5-氟尿嘧啶、替尼泊苷、莫立替尼、埃博霉素、长春瑞滨、放线菌素D、米托蒽醌、丝裂霉素、博来霉素、伊立替康等含羟基或氨基的药物)、或通过多巴胺与药物中硼酸基团反应形成pH敏感硼酸脂(涉及药物硼替佐米等含硼酸基团的药物)、或通过固相合成直接形成酰胺键(涉及药物p53激活肽、抗菌肽、多肽毒素等多肽药物)的多肽-药物复合物。
另一方面，本发明提供了一种前述稳定化A7R多肽复合物的制备方法，所述方法包括：
当所述抗肿瘤药物为含酮或醛基的药物时，前述的稳定化A7R多肽通过pH敏感腙键与所述抗肿瘤药物连接制备所述稳定化A7R多肽复合物；
当所述抗肿瘤药物为含羟基或氨基的药物时，前述的稳定化A7R多肽通过二硫键与所述抗肿瘤药物连接制备所述稳定化A7R多肽复合物；
当所述抗肿瘤药物为含硼酸基团的药物时，前述的稳定化A7R多肽通过pH敏感的硼酸脂键与所述抗肿瘤药物连接制备所述稳定化A7R多肽复合物；和/或
当所述抗肿瘤药物为多肽药物时，前述的稳定化A7R多肽与所述抗肿瘤药物缩合制备所述稳定化A7R多肽复合物。
又一方面，本发明提供了一种稳定化A7R多肽复合物，所述稳定化A7R多肽复合物为前述的稳定化A7R多肽修饰高分子载体材料，其中，所述稳定化A7R多肽复合物的结构为DA7R-聚乙二醇-Z和/或cA7R-聚乙二醇-Z，Z为所述高分子载体材料。
优选地，所述高分子载体材料选自磷脂、聚乳酸、乳酸羟基乙酸共聚物和聚已内酯中的一种或多种。
具体地，本发明所设计的cA7R和连接半胱氨酸后的DA7R，可修饰在含马来酰亚胺功能基的聚乙二醇-二硬脂酰基磷脂酰乙醇胺(PEG-DSPE)、 聚乙二醇-聚乳酸(PEG-PLA)、聚乙二醇-乳酸羟基乙酸共聚物(PEG-PLGA)、聚乙二醇-聚己内酯(PEG-PCL)等高分子载体材料上，可用于DA7R或cA7R修饰的脂质体、聚合物胶束、聚合物圆盘、纳米粒等纳米递药系统的构建。
再一方面，本发明提供了一种前述稳定化A7R多肽复合物的制备方法，所述方法包括：前述的稳定化A7R多肽或巯基化的前述稳定化A7R多肽与马来酰亚胺-聚乙二醇-高分子载体材料反应制备所述稳定化A7R多肽复合物。
另一方面，本发明提供了一种递药系统，所述递药系统包括前述的稳定化A7R多肽复合物。优选地，所述递药系统为脂质体递药系统、聚合物胶束递药系统、聚合物圆盘递药系统或纳米粒递药系统。
作为优选实施方式，本发明还提供了还包括所述稳定化A7R多肽复合物以外的(1)影像物质和/或(2)抗肿瘤药物的前述递药系统。
优选地，所述(1)影像物质选自荧光物质、近红外染料和磁共振影像剂中的一种或多种。更优选地，所述荧光物质为5-羧基荧光素(5-FAM)，所述近红外染料选自Cy5.5、IR820、DiR和DiD中的一种或多种，和/或磁共振影像剂为Gd-DTPA。和/或所述(2)抗肿瘤药物选自阿霉素、表阿霉素、紫杉醇、多烯紫杉醇、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、硼替佐米、卡非佐米、环磷酰胺、依托泊苷、吉西他滨、阿糖胞苷、5-氟尿嘧啶、替尼泊苷、莫立替尼、埃博霉素、长春瑞滨、放线菌素D、米托蒽醌、丝裂霉素、博来霉素、伊立替康、顺铂、奥沙利铂、p53激活肽、蜂毒肽、蝎毒肽、贝伐单抗和曲妥单抗中的一种或多种。
具体地，本发明所设计的DA7R或cA7R修饰的纳米递药系统可包载阿霉素、表阿霉素、紫杉醇、多烯紫杉醇、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、硼替佐米、卡非佐米、环磷酰胺、依托泊苷、吉西他滨、阿糖胞苷、5-氟尿嘧啶、替尼泊苷、莫立替尼、埃博霉素、长春瑞滨、放线菌素D、米托蒽醌、丝裂霉素、博来霉素、伊立替康、顺铂、奥沙利铂、p53激活肽、蜂毒肽、蝎毒肽、贝伐单抗、曲妥单抗等；也可包载荧光物质、近红外染料和磁共振影像剂，如FAM、Cy5.5、IR820、DiR、DiD、Gd-DTPA等。
又一方面，本发明提供了前述的稳定化A7R多肽、前述的稳定化A7R多肽复合物、前述的递药系统在制备用于诊断、示踪和/或治疗肿瘤的药品或医疗产品中的应用。
优选地：
所述肿瘤为高表达新生血管内皮生长因子受体2肿瘤或高表达神经纤毛蛋白-1肿瘤。
具体地，本发明所设计的DA7R或cA7R具有抗肿瘤作用，进一步可与抗肿瘤药物联合给药、协同治疗，这些药物包括阿霉素、表阿霉素、紫杉醇、多烯紫杉醇、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、硼替佐米、卡非佐米、环磷酰胺、替莫唑胺、甲氨蝶呤、依托泊苷、巯嘌呤、吉西他滨、阿糖胞苷、5-氟尿嘧啶、替尼泊苷、莫立替尼、埃博霉素、长春瑞滨、放线菌素D、米托蒽醌、丝裂霉素、博来霉素、伊立替康、顺铂、奥沙利铂、p53 激活肽、蜂毒肽、蝎毒肽、贝伐单抗、曲妥单抗等。
本发明所设计的DA7R和cA7R可介导药物或纳米递药系统靶向血管内皮生长因子受体2(VEGFR2)和神经纤毛蛋白-1(NRP-1)高表达的细胞及其组织，用于肿瘤的靶向诊断和治疗；可通过与VEGFR2结合发挥抗肿瘤作用，同时，通过靶向NRP-1，可增加药物对肿瘤组织的穿透力，进一步用于抗肿瘤药物的协同增效。
再一方面，本发明还提供了一种用于诊断、示踪和/或治疗肿瘤的组合产品，所述组合产品包括选自以下的一种或多种成分：前述的稳定化A7R多肽、前述的稳定化A7R多肽复合物和前述的递药系统。
优选地，所述组合产品为试剂盒，和/或
所述肿瘤为高表达新生血管内皮生长因子受体2肿瘤或高表达神经纤毛蛋白-1肿瘤。
另一方面，本发明还提供了一种诊断、示踪和/或治疗肿瘤的方法，包括对患有所述肿瘤或疑患有所述肿瘤的患者通过口服或非口服途径给予有效剂量的选自以下的一种或多种物质：前述的稳定化A7R多肽、前述的稳定化A7R多肽复合物、前述的递药系统和前述的组合产品。
优选地，所述肿瘤为高表达新生血管内皮生长因子受体2肿瘤或高表达神经纤毛蛋白-1肿瘤；和/或
优选地，当所述方法用于治疗肿瘤时，所述方法包括对患有所述肿瘤或疑患有所述肿瘤的患者通过口服或非口服途径给予有效剂量的(1)前述的稳定化A7R多肽与(2)一种或多种其他抗肿瘤药物，所述其他抗肿瘤药物优选选自：阿霉素、表阿霉素、紫杉醇、多烯紫杉醇、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、硼替佐米、卡非佐米、环磷酰胺、替莫唑胺、依托泊苷、巯嘌呤、吉西他滨、阿糖胞苷、5-氟尿嘧啶、替尼泊苷、莫立替尼、埃博霉素、长春瑞滨、放线菌素D、米托蒽醌、丝裂霉素、博来霉素、伊立替康、顺铂、奥沙利铂、p53激活肽、蜂毒肽、蝎毒肽、贝伐单抗和曲妥单抗中的一种或多种。
所述口服或非口服途径可以为通过口服、注射、贴片、喷雾和其他已知的一种或多种递送给所述患者。所述有效量可以包括对治疗、降低、缓和、减轻、消除或状况的一种或多种症状有效的量，所述状况寻求被治疗，或可选地，所述状况寻求被避免，或另外在所述状况或其效果中产生临床上可确认的有利变化。
又一方面，本发明还提供了前述的稳定化A7R多肽作为抗肿瘤活性成分在制备用于抗肿瘤的药品和/或医疗产品中的应用。。
再一方面，本发明还提供了前述的稳定化A7R多肽在制备肿瘤靶向产品中的应用。优选地：所述肿瘤靶向产品用于靶向VEGFR2和NRP-1高表达的肿瘤新生血管、拟态血管和肿瘤细胞；和/或所述肿瘤靶向产品为用于诊断、示踪和/或治疗肿瘤的药品、实验试剂和/或医疗产品。
另一方面，本发明还提供了前述的稳定化A7R多肽在制备用于协同增 效其他抗肿瘤药物疗效的药品、实验试剂和/或医疗产品中的应用；
优选地，所述其他抗肿瘤药物优选选自：阿霉素、表阿霉素、紫杉醇、多烯紫杉醇、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、硼替佐米、卡非佐米、环磷酰胺、替莫唑胺、依托泊苷、巯嘌呤、吉西他滨、阿糖胞苷、5-氟尿嘧啶、替尼泊苷、莫立替尼、埃博霉素、长春瑞滨、放线菌素D、米托蒽醌、丝裂霉素、博来霉素、伊立替康、顺铂、奥沙利铂、p53激活肽、蜂毒肽、蝎毒肽、贝伐单抗和曲妥单抗中的一种或多种。
本发明提供了高度稳定且可同时靶向血管内皮生长因子受体2和神经纤毛蛋白-1高表达细胞的逆序D构型多肽和首尾酰胺键环合多肽，并构建其复合物和修饰的纳米递药系统，实现肿瘤的靶向诊断和治疗；利用A7R多肽与VEGFR2高结合的特点，起到抑制肿瘤血管生成而达到抗肿瘤的作用，同时，A7R多肽还具有CendR多肽类似结构靶向NRP-1，可增加药物对肿瘤组织的穿透力，因而进一步与抗肿瘤药物联用可协同增效。
具体地，本发明提供了DA7R、cA7R制备和性质考察以及上述所修饰的药物复合物和纳米递药系统用于肿瘤诊疗的物质基础。一种具体的实施方案可以如下：
1.DA7R、DA7R-Cys及其荧光标记物(DA7R-Fluorescein)的合成
根据逆序多肽合成技术，采用固相合成方法制备DA7R和DA7R-Cys。通过马来酰亚胺基团与巯基的Michael加成反应合成了DA7R-Fluorescein。HPLC、MS表征结构。
2.cA7R及其荧光标记物(cA7R-Fluorescein)的合成
根据“Native Chemical Ligation”反应制备cA7R。通过马来酰亚胺基团与巯基的Michael加成反应合成了cA7R-Fluorescein。HPLC、MS表征结构。3.DA7R和cA7R稳定性和受体亲和性评价
从血清稳定性、与血管内皮生长因子受体2(VEGFR2)和神经纤毛蛋白-1(NRP-1)结合能力和与高表达这两种受体的细胞摄取能力三方面进行DA7R和cA7R性质的考察。将DA7R、cA7R和LA7R分别与小鼠血清在37℃进行孵育，在不同时间点检测多肽的浓度进行稳定性的比较。采用表面等离子共振法评价DA7R、cA7R和LA7R与两种受体蛋白的结合能力。比较
DA7R-Fluorescein、cA7R-Fluorescein、LA7R-Fluorescein对血管内皮生长因子受体2和神经纤毛蛋白-1蛋白高表达的细胞(如：脐静脉内皮细胞HUVEC)和模型肿瘤细胞(如：脑胶质瘤细胞U87)的体外靶向性。比较体外肿瘤拟态血管模型对DA7R-Fluorescein、cA7R-Fluorescein、LA7R-Fluorescein的摄取能力。
4.DA7R和cA7R对药物修饰
cA7R和连接半胱氨酸后的DA7R与药物的马来酰亚胺己肼衍生物反应，形成含pH敏感腙键的多肽-药物复合物，其中所涉及药物包括阿霉素、表阿霉素等含酮或醛基的药物。
cA7R和连接半胱氨酸后的DA7R与药物的3-(2-吡啶二巯基)丙酸衍生物反应，形成含二硫键的多肽-药物复合物，其中所涉及药物包括紫杉醇、多烯紫杉醇、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱等含羟基或氨基的药物。
cA7R和DA7R通过修饰上多巴胺进而与药物的硼酸基团反应，形成含pH敏感硼酸脂的多肽-药物复合物，其中所涉及药物包括硼替佐米等含硼酸基团的药物。
cA7R和DA7R通过固相合成直接与多肽药物缩合制成融合多肽，其中所涉及药物包括p53激活肽、抗菌肽、多肽毒素等多肽药物。
5.A7R-药物复合物靶向性、稳定性与药效学评价
考察U87细胞和HUVEC细胞对DA7R-阿霉素复合物(DA7R-Aldoxorubicin)和LA7R-阿霉素复合物(LA7R-Aldoxorubicin)的摄取情况。
将DA7R-Aldoxorubicin和LA7R-Aldoxorubicin分别与不同pH的0.1M磷酸盐缓冲液37℃孵育，在不同时间点检测A7R-Aldoxorubicin的浓度进行pH稳定性的比较。将DA7R-Aldoxorubicin和LA7R-Aldoxorubicin分别与小鼠血清在37℃进行孵育，在不同时间点检测A7R-Aldoxorubicin的浓度进行血清稳定性的比较。
通过荷U87皮下移植瘤模型裸鼠尾静脉分别注射DA7R-Aldoxorubicin、LA7R-Aldoxorubicin、Aldoxorubicin和游离阿霉素，1h后取出肿瘤制作冰冻切片，CD31标记血管，DAPI染核，比较各组药物在肿瘤内分布。
考察DA7R-Aldoxorubicin、LA7R-Aldoxorubicin、Aldoxorubicin和游离阿霉素对U87细胞和HUVEC细胞生长的抑制。
荷U87皮下移植瘤模型裸鼠尾静脉分别注射DA7R-Aldoxorubicin、LA7R-Aldoxorubicin、Aldoxorubicin、游离阿霉素和生理盐水，以瘤体积、瘤重为指标评价体内抗肿瘤效果。
将皮下瘤药效学试验的各给药组小鼠的脏器组织解剖后固定于4％多聚甲醛的PBS溶液中，石蜡包埋切片进行HE染色，考察各组药物的全身毒性。
荷U87皮下移植瘤模型裸鼠尾静脉分别注射不同剂量DA7R-Aldoxorubicin、游离阿霉素，多肽DA7R和生理盐水，以瘤体积、瘤重为指标评价体内抗肿瘤效果。
6.DA7R-PEG-脂质体递药系统和cA7R-PEG-脂质体递药系统的构建与表征
首先合成DA7R、cA7R、LA7R修饰的高分子材料DA7R-PEG-DSPE、cA7R-PEG-DSPE和LA7R-PEG-DSPE。将DA7R-Cys与Mal-PEG-DSPE在pH7.2的PBS和DMF的混合溶液中反应得到DA7R-PEG-DSPE。将cA7R、LA7R-Cys分别与Mal-PEG-DSPE按上述方法反应得到cA7R-PEG-DSPE和LA7R-PEG-DSPE。
然后分别制备DA7R、cA7R、LA7R修饰的脂质体(DA7R-PEG-脂质体、cA7R-PEG-脂质体和LA7R-PEG-脂质体)。以一定比例的HSPC/Chol/mPEG2000-DSPE/DA7R-PEG-DSPE或cA7R-PEG-DSPE或LA7R-PEG-DSPE为膜材料，采用成膜水化法制备脂质体，用挤压过膜的方法减小脂质体粒径，并分别包载DiR、FAM、阿霉素(DOX)等药物，构建平均粒径在100nm左右的脂质体。动态光散射法测定粒径分布，负染色电镜法观察脂质体形态。
7.DA7R-PEG-脂质体递药系统和cA7R-PEG-脂质体递药系统的体内外肿瘤靶向性评价
考察U87细胞、HUVEC细胞和U87拟态血管体外模型对DA7R-PEG-脂质体/FAM、cA7R-PEG-脂质体/FAM、LA7R-PEG-脂质体/FAM和mPEG- 脂质体/FAM的摄取情况。
通过荷U87皮下移植瘤模型裸鼠尾静脉分别注射DA7R-PEG-脂质体/DiR、cA7R-PEG-脂质体/DiR、LA7R-PEG-脂质体/DiR和mPEG-脂质体/DiR，比较不同递药系统在各时间点的肿瘤内分布。
8.DA7R-PEG-脂质体递药系统和cA7R-PEG-脂质体递药系统的体内抗肿瘤效果评价
通过荷U87皮下移植瘤模型裸鼠尾静脉分别注射DA7R-PEG-脂质体/阿霉素、cA7R-PEG-脂质体/阿霉素、LA7R-PEG-脂质体/阿霉素、mPEG-脂质体/阿霉素、游离阿霉素和生理盐水，以瘤体积、瘤重和肿瘤组织细胞凋亡、新生血管和拟态血管数量为指标评价不同载阿霉素递药系统的体内抗肿瘤效果。
9.A7R及A7R与抗肿瘤药物联合用药的靶向性、药效学评价
荷U87皮下移植瘤模型裸鼠尾静脉分别注射DA7R、LA7R、DA7R与DOX、LA7R与DOX、DOX以及生理盐水，以瘤体积、瘤重为指标评价体内抗肿瘤效果。
通过荷U87原位移植瘤模型裸鼠尾静脉分别注射DA7R、DA7R与DOX、DA7R与阿霉素脂质体(LS/DOX)、DA7R与替莫唑胺(TMZ)、DOX、LS/DOX、TMZ以及生理盐水，以中位生存期为指标评价抗肿瘤效果。
荷A549皮下移植瘤模型裸鼠尾静脉分别注射DA7R、DA7R与DOX、DA7R与LS/DOX、DOX、LS/DOX以及生理盐水，以瘤体积、瘤重为指标评价体内抗肿瘤效果。
通过荷U87皮下移植瘤模型裸鼠尾静脉分别注射DA7R与DOX、LA7R与DOX和游离阿霉素，1h后取出肿瘤制作冰冻切片，CD31标记血管，DAPI染核，比较各组药物在肿瘤内分布。
10.A7R、A7R抗肿瘤药物复合物及A7R与抗肿瘤药物联合用药的最大耐受剂量
正常小鼠尾静脉分别注射DA7R、DA7R-Aldoxorubicin、DA7R与DOX、Aldoxorubicin、DOX，考察最大耐受剂量(MTD)。
本发明的试验结果表明：DA7R和cA7R比LA7R在血清中具有更高的稳定性，且与VEGFR2和NRP-1受体蛋白的亲和性相近，与高表达这两种受体的模型细胞亲和活性相近，但在模型动物体内具有更好的肿瘤组织靶向能力和影像效果；与LA7R修饰的药物复合物或纳米递药系统相比，DA7R或cA7R修饰的药物复合物或纳米递药系统均显示出了更好的肿瘤靶向性能和更强的抗肿瘤效果。
本发明提供了cA7R和DA7R，具有明显的抗肿瘤活性，显著优于LA7R。并且作为协同增效剂，与抗肿瘤药物联合用药，发挥更强的抗肿瘤效果。
下面通过附图和实施例，对本发明的技术方案做进一步的详细描述。
附图的简要说明
图1示出了DA7R的ESI-MS图谱；
图2示出了DA7R-Cys的ESI-MS图谱；
图3示出了LA7R的ESI-MS图谱；
图4示出了LA7R-Cys的ESI-MS图谱；
图5示出了cA7R的ESI-MS图谱；
图6示出了DA7R-Fluorescein的ESI-MS图谱；
图7示出了LA7R-Fluorescein的ESI-MS图谱；
图8示出了cA7R-Fluorescein的ESI-MS图谱；
图9示出了DA7R-Aldoxorubicin的ESI-MS图谱；
图10示出了LA7R-Aldoxorubicin的ESI-MS图谱；
图11示出了DA7R-PEG3400-DSPE和cA7R-PEG3400-DSPE的1H-NMR图谱；
图12示出了载阿霉素脂质体的粒径和电镜照片，
图A和图B分别为各阿霉素脂质体的粒径和电镜照片；
图13示出了DA7R和cA7R的血清稳定性；
图14示出了DA7R和cA7R与VEGFR2结合活性；
图15示出了DA7R及cA7R与NRP-1结合活性；
图16示出了脑胶质瘤细胞U87对Fluorescein标记多肽的摄取，
图A和图B分别为Fluorescein标记的DA7R、cA7R和LA7R与U87细胞作用4h后的激光共聚焦照片和流式细胞荧光检测结果；
图17示出了脐静脉内皮细胞HUVEC对Fluorescein标记多肽的摄取，
图A和图B分别为Fluorescein标记的DA7R、cA7R和LA7R与HUVEC细胞作用4h后的激光共聚焦照片和流式细胞荧光检测结果；
图18示出了U87拟态血管体外模型对Fluorescein标记多肽的摄取；
图19示出了Fluorescein标记多肽的皮下移植瘤内分布，
图A为荷U87皮下移植瘤裸鼠尾静脉注射Fluorescein标记多肽1h后的离体肿瘤影像分布结果；图B为离体脏器的影像分布结果；图C为离体肿瘤的荧光半定量结果；图D为离体肿瘤和脏器的荧光半定量结果；
图20示出了脑胶质瘤细胞U87对A7R-Aldoxorubicin的摄取，
图A和图B分别为A7R-Aldoxorubicin与U87细胞作用4h后的激光共聚焦照片和流式细胞荧光检测结果；
图21示出了脐静脉内皮细胞HUVEC对A7R-Aldoxorubicin的摄取，
图A和图B分别为A7R-Aldoxorubicin与HUVEC细胞作用4h后的激光共聚焦照片和流式细胞荧光检测结果；
图22示出了DA7R-Aldoxorubicin和cA7R-Aldoxorubicin的pH稳定性；
图23示出了DA7R-Aldoxorubicin和cA7R-Aldoxorubicin的血清稳定性；
图24示出了A7R-Aldoxorubicin皮下瘤内分布；
图25示出了A7R-Aldoxorubicin体外抗U87细胞和HUVEC细胞活性曲线，
图A和图B分别为DA7R-Aldoxorubicin、LA7R-Aldoxorubicin、Aldoxorubicin和DOX抗U87细胞和HUVEC细胞的活性曲线；
图26示出了A7R-Aldoxorubicin皮下瘤抑制实验，
图A为各组裸鼠肿瘤体积随时间变化的曲线，图B为各组裸鼠体重随时间变化的曲线，图C为将裸鼠处死取出肿瘤组织后称重的统计分析结果；
图27示出了不同剂量A7R-Aldoxorubicin的U87皮下瘤抑制实验，
图A为各组裸鼠肿瘤体积随时间变化的曲线，图B为各组裸鼠体重随时间变化的曲线，图C为将裸鼠处死取出肿瘤组织后称重的统计分析结果；
图28示出了CD31/PAS染色和TUNEL染色结果；
图29示出了不同剂量A7R-Aldoxorubicin的U87皮下瘤抑制免疫组化试验，
图A为不同剂量DA7R-Aldoxorubicin和DOX及多肽DA7R抑制新生血管形成的CD31/PAS双染色照片(bar＝100μm)，其中新生血管细胞呈棕黄色或棕褐色，图B为不同剂量DA7R-Aldoxorubicin和DOX及多肽DA7R促进皮下瘤凋亡的TUNEL染色照片(bar＝100μm)，其中凋亡的阳性细胞核呈棕黄色或棕褐色；
图30示出了A7R-Aldoxorubicin的全身毒性；
图31示出了U87细胞对包载5-FAM多肽修饰脂质体的摄取，
图A和图B分别为包载荧光素5-FAM的DA7R、cA7R和LA7R修饰脂质体与U87细胞作用4h后的激光共聚焦照片和流式细胞荧光检测结果；
图32示出了HUVEC细胞对包载5-FAM脂质体的摄取；
图A和图B分别为包载5-FAM的各处方脂质体于37℃分别与HUVEC细胞作用4h后的激光共聚焦照片和流式结果；
图33示出了U87拟态血管体外模型对包载5-FAM脂质体的摄取；
图34示出了载近红外染料的PEG-脂质体的皮下瘤内分布，
图A为尾静脉注射24小时后的在体荧光分布图像，图B为给药后各个时间点肿瘤内荧光强度半定量结果，图C为脏器的荧光分布图像，图D为图C的荧光半定量统计结果；
图35示出了载阿霉素脂质体体外抗U87细胞和HUVEC细胞活性曲线，
图A和图B分别为DA7R-LS/DOX、cA7R-LS/DOX、LA7R-LS/DOX、LS/DOX和DOX抗U87细胞和HUVEC细胞的活性曲线；
图36示出了载阿霉素脂质体体外对新生血管形成的抑制，
图A为DA7R-LS/DOX、cA7R-LS/DOX、LA7R-LS/DOX、LS/DOX和DOX对新生血管体外模型的抑制照片，图B为各组血管样结构的形成率统计结果；
图37示出了载阿霉素脂质体体外对拟态血管形成的抑制，
图A为DA7R-LS/DOX、cA7R-LS/DOX、LA7R-LS/DOX、LS/DOX和DOX对拟态血管体外模型的抑制照片，图B为各组拟态血管结构的形成率统计结果；
图38示出了载阿霉素脂质体皮下瘤抑制实验，
图A为各组裸鼠肿瘤体积随时间变化的曲线，图B为将裸鼠处死取出肿瘤组织后称重的统计分析结果；
图39示出了TUNEL染色结果，
图A为DA7R-LS/DOX、cA7R-LS/DOX、LA7R-LS/DOX、LS/DOX和DOX促进皮下瘤凋亡的TUNEL染色照片(bar＝50μm)，其中凋亡的阳性细胞核呈棕黄色或棕褐色，图B为阳性细胞数的统计结果；
图40示出了CD31/PAS双染色结果，
图A为DA7R-LS/DOX、cA7R-LS/DOX、LA7R-LS/DOX、LS/DOX和DOX抑制新生血管形成的CD31/PAS双染色照片(bar＝100μm)，其中新生血管细胞核呈棕黄色或棕褐色，图B为新生血管数的统计结果；
图41示出了A7R及A7R与DOX联合给药皮下瘤抑制试验，
图A为各组裸鼠肿瘤体积随时间变化的曲线，图B为各组裸鼠体重随时间变化的曲线，图C为将裸鼠处死取出肿瘤组织后称重的统计分析结果；图42示出了DA7R及DA7R与DOX联合给药抗U87原位瘤药效学试验；
图43示出了DA7R与LS/DOX联合给药抗U87原位瘤药效学试验；
图44示出了DA7R及DA7R与TMZ联合用药的药效学试验；
图45示出了DA7R及DA7R与DOX联合给药抗A549皮下瘤药效学试验；
图46示出了A7R与DOX联合给药U87皮下瘤内分布；
图47示出了最大耐受剂量(MTD)试验，
图A为不同剂量下DOX组小鼠体重变化曲线，图B为不同剂量下Aldoxorubicin组小鼠体重变化曲线，图C为不同剂量下DA7R组小鼠体重变化曲线，图D为不同剂量下DA7R与DOX联合给药组小鼠体重变化曲线，图E为不同剂量下DA7R-Aldoxorubicin组小鼠体重变化曲线，图F为第8天各组体重变化条形统计图。
实施发明的最佳方式
下面通过具体的实施例进一步说明本发明，但是，应当理解为，这些实施例仅仅是用于更详细具体地说明之用，而不应理解为用于以任何形式限制本发明。
本部分对本发明试验中所使用到的材料以及试验方法进行一般性的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的，但是本发明仍然在此作尽可能详细描述。本领域技术人员清楚，在上下文中，如果未特别说明，本发明所用材料和操作方法是本领域公知的。
实施例1：LA7R和DA7R、LA7R-Cys和DA7R-Cys的合成与表征
采用逆序固相多肽合成法，设计并合成由非天然D构型氨基酸所构成的DA7R(序列为DRDPDPDLDWDTDA)和DA7R-Cys(序列为DCDRDPDPDLDWDTDA)。合成L构型氨基酸所构成的LA7R(序列为ATWLPPR)和LA7R-Cys(序列为ATWLPPRC)。
具体方法：将PAM-Boc树脂用三氟乙酸(TFA)脱保护。用Boc保护氨基酸依次反应，反应完成后，三氟乙酸脱Boc保护后，依次用DMF、DCM/MeOH(1:1，v/v)洗涤树脂，真空干燥。将树脂放入多肽切割管中，加入适量P-cresol，然后通入氟化氢，冰浴搅拌反应1h。反应结束后减压抽去管中氟化氢，冰乙醚洗涤沉淀3次，残余沉淀以20％乙腈溶解，收集滤液后旋蒸，得到多肽粗品溶液。多肽粗品用乙腈/水(含0.1％TFA)体系分离纯化。
ESI-MS表征DA7R、DA7R-Cys、LA7R及LA7R-Cys的纯度和分子量(Mw)，分别为840.4、941.8、840.4和941.8，均与理论分子量相符合。质谱图见附图1、2、3、4。
实施例2：cA7R的合成与表征
先采用固态多肽合成法，设计并合成直链多肽A7R-Mpr-Leu。然后采用“Native Chemical Ligation”反应合成目标产物cA7R。将纯化的A7R-Mpr-Leu溶解于6M盐酸胍溶液中，浓度为1mg/mL，加入1‰(v/v)的催化剂硫代苯酚，密闭搅拌反应，用HPLC检测反应进程，待原料峰消失，产物峰高不再增加时加入20％哌啶以脱去终止反应。反应结束后，反应液稀释2-3倍，用0.22μm滤膜过滤，用制备HPLC分离纯化得到cA7R。ESI-MS表征cA7R的纯度和分子量(Mw)，为924.8，与理论分子量相符合。质谱图见附图5。
实施例3：A7R-Fluorescein的合成与表征
将实施例1或实施例2得到的DA7R-Cys、LA7R-Cys或cA7R溶于0.1M的PBS溶液中(pH7.2)，取Fluorescein-5-maleimide溶于DMF，两者混合后磁力搅拌反应，HPLC监测，待DA7R-Cys、LA7R-Cys或cA7R反应完全后停止反应，制备液相纯化，用乙腈/水(含0.1％TFA)体系分离纯化。冷冻干燥得DA7R-Fluorescein、LA7R-Fluorescein或cA7R-Fluorescein纯品。
ESI-MS表征DA7R-Fluorescein、LA7R-Fluorescein或cA7R-Fluorescein，分别为1368.0、1368.0和1351.6，均与理论分子量相符合。质谱图见附图6、7、8。
实施例4：A7R-DTPA-Gd的制备
maleimide-DTPA溶于DMF，同上与DA7R-Cys、LA7R-Cys或cA7R溶于的PBS溶液混合搅拌反应，制备液相纯化，冷冻干燥得DA7R-DTPA、LA7R-DTPA或cA7R-DTPA纯品，螯合Gd即得A7R-DTPA-Gd。
实施例5：A7R-药物复合物的制备
以A7R-阿霉素偶联物制备作为A7R连接含酮或醛基药物的实施例。9.4mg含巯基的A7R多肽溶于3mL磷酸盐缓冲液(0.1mM，pH 7.0)，于4℃搅拌20min。然后加入4倍摩尔量的阿霉素6-马来酰亚胺己肼衍生物，于室温避光反应1h。反应液用C18制备柱进行分离(色谱柱：waters X bridge19×300mm；流动相：A 0.01M pH7.4磷酸盐缓冲液，B乙腈；洗脱方法：100％A～100％B线性梯度)，收集相应组分，脱盐后冷冻干燥得DA7R或LA7R-阿霉素偶联物(即DA7R-Aldoxorubicin或LA7R-Aldoxorubicin)。质谱图见附图9、10，ESI-MS均为1692.6，均与理论分子量相符合。
以A7R-紫杉醇复合物作为A7R以二硫键连接药物的实施例。200mg紫杉醇溶于10mL氯仿中，冷却至0-5℃，先后加入39.99mg DCC及60.4mg 3-(2-吡啶二巯基)丙酸，加料完毕后，升至室温反应过夜。反应液过滤，经柱层析纯化(CHCl3/MeOH＝50:1-15:1，V/V洗脱)得紫杉醇3-(2-吡啶二巯基)丙酸衍生物。紫杉醇3-(2-吡啶二巯基)丙酸衍生物溶解在5mL DMF中，1.5倍摩尔量的含巯基的A7R溶解在PBS/DMF中，溶液pH值保持4～5将紫杉醇3-(2-吡啶二巯基)丙酸衍生物滴加至巯基多肽溶液中，于室温反应6h，经制备液相制备冻干得A7R-紫杉醇复合物。
以A7R-硼替佐咪复合物作为A7R氮端修饰药物的实施例。依照A7R的合成在树脂上依次接入氨基酸，待多肽的所有氨基酸残基接入完毕，三氟乙酸脱去氮端的Boc保护。加入含3倍摩尔量的丁二酸酐与DIEA的DMF溶液，于室温反应30min。洗涤树脂后，加入5倍摩尔量的三甲基氯硅烷保护多巴胺，并以HBTU/DIEA为缩合剂，于室温反应1h。树脂用HF切割，并经制备型HPLC纯化得多肽-多巴胺衍生物。在pH7.4的缓冲液中，多肽-多巴胺衍生物与硼替佐咪以摩尔比1:1混合即得A7R-硼替佐咪复合物。
以A7R-p53激活肽PMI复合物作为A7R融合多肽药物的实施例。直接通过固相多肽合成法制得，具体方法为：确定A7R-PMI多肽序列后，按与制备A7R相同的方法依次接入氨基酸，经HF切割并纯化后得A7R-PMI融合多肽。
实施例7：A7R-PEG-DSPE的合成与表征
将DA7R-Cys、LA7R-Cys或cA7R溶于0.1M的PBS溶液中(pH7.2)，取Mal-PEG-DSPE溶于DMF，两者混合后磁力搅拌反应，HPLC监测，待Mal-PEG-DSPE反应完全后停止反应，过量的DA7R-Cys、LA7R-Cys、cA7R和DMF透析(截留分子量3.5kDa)除去，冷冻干燥得DA7R-PEG-DSPE、LA7R-PEG-DSPE或cA7R-PEG-DSPE，NMR表征其结构(见附图11)。Mal-PEG-DSPE的核磁图谱于6.7ppm显示出马来酰亚胺峰，而DA7R-PEG-DSPE、LA7R-PEG-DSPE与cA7R-PEG-DSPE的核磁图谱中该峰消失，显示Mal-PEG-DSPE中的马来酰亚胺基团已连接上A7R。
实施例8：A7R-PEG-脂质体的制备与表征
A7R-PEG-脂质体膜材料处方为HSPC/Chol/mPEG2000-DSPE/A7R-PEG-DSPE(52:43:3:2，mol/mol)，针对不同性质的药物制备过程略有不同。
具体方法：
水溶性药物：称取上述膜材料溶于氯仿，减压旋转蒸发除去有机溶媒，得均匀脂质膜，真空干燥24h。加入水溶性荧光素(5-FAM)或核磁影像剂(Gd-DTPA)水溶液水化，60℃水浴震荡2h，得脂质体混悬液。在60℃温度下，使用高压均质机(若脂质体体积少于10mL则改用微型挤出器)依次将脂质体挤压过400、200、100和50nm核孔膜，使其粒径减小。然后以生理盐水为洗脱液过葡聚糖凝胶G-50柱分离除去未包封的5-FAM或Gd-DTPA，得到包载5-FAM或Gd-DTPA的脂质体。
疏水性药物：膜材料及疏水性近红外染料(DiR)溶于氯仿，减压旋转蒸发除去有机溶媒，得均匀脂质膜，真空干燥24h。加入生理盐水溶液水化，60℃水浴震荡2h，得脂质体混悬液。后处理同上得到包载DiR的脂质体。
弱酸弱碱性药物：采用主动载药法。如阿霉素脂质体，通过硫酸铵梯度法制备。动态光散射法测定粒径分布(附图12A)，负染色电镜法观察脂质体形态(见附图12B)。由图可见，A7R修饰脂质体与未修饰脂质体大小和形态均无显著差异。
试验例1：A7R的血清稳定性考察
将DA7R、cA7R及LA7R配成1mg/mL水溶液，取0.1mL加入0.9mL的25％小鼠血清中，37℃孵育，分别于0和15min，0.5、1、2和4h取出100μL反应液，加入20μL乙腈沉淀血清中蛋白，4℃静置20min，12000转/分钟离心10min，取上清液20μL进行HPLC分析。血清稳定性结果见附图13。图纵坐标为完整多肽的残留百分比，可见DA7R和cA7R在25％小鼠血清中的稳定性显著高于LA7R，孵育2小时，LA7R完全降解、cA7R降解不足35％、DA7R几乎不降解。结果表明，DA7R与cA7R具有比LA7R更好的血清稳定性。
试验例2：A7R与血管内皮生长因子受体2(VEGFR2)的结合活性实验
通过biacore系统进行预结合分析，选取pH4.5为最佳VEGFR2蛋白与CM5芯片结合pH。将重组人VEGFR2蛋白偶联至CM5芯片上，RU值达到 目标值。将DA7R、cA7R及LA7R分别配置成浓度为5、10、20、40、80和160nM的样品溶液。从低到高依次进样，用Biacore T200Evaluation software软件分析DA7R、cA7R及LA7R与VEGFR2蛋白的结合活性，并分别计算其KD值(附图14)，可见DA7R、cA7R和LA7R与VEGFR2蛋白的结合活性相似，KD值分别为8.414nM、6.794nM和9.289nM。
试验例3：A7R与神经纤毛蛋白-1(NRP-1)的结合活性实验
通过biacore系统进行预结合分析，选取pH4.5为最佳NRP-1蛋白与CM5芯片结合pH。将重组人NRP-1蛋白偶联至CM5芯片上，RU值达到目标值。将DA7R、cA7R及LA7R分别配置成浓度为2.5、5、10、20、40和80nM的样品溶液。从低到高依次进样，用Biacore T200Evaluation software软件分析DA7R、cA7R及LA7R与NRP-1蛋白的结合活性，并分别计算其KD值(附图15)，可见DA7R、cA7R和LA7R与NRP-1蛋白的结合活性相似，KD值分别为2.31nM、10.57nM和6.62nM。
试验例4：A7R对脑胶质瘤细胞U87的体外靶向性
取对数生长期的单层培养的脑胶质瘤细胞(U87细胞)，用0.25％胰蛋白酶消化单层培养细胞，用含10％胎牛血清的DMEM培养液配成单细胞悬液，以每孔1×105个细胞接种于12孔培养板中，每孔体积1mL，将培养板移入二氧化碳培养箱中，37℃、5％CO2及饱和湿度条件下培养24h后，用含10％胎牛血清的DMEM培养液配制浓度为5μM的FAM、DA7R-Fluorescein、cA7R-Fluorescein及LA7R-Fluorescein溶液。将培养板中的培养液吸出，分别加入上述溶液，37℃孵育4h，吸弃上清液。用PBS溶液洗三次，甲醛固定液固定细胞，DAPI进行细胞核染色后，激光共聚焦观察，细胞内化照片见附图16A。另用PBS洗三次后，进行流式细胞仪分析，结果见附图16B。可见U87细胞对DA7R、cA7R和LA7R的摄取明显高于游离荧光素，但对三种多肽的摄取没有明显差异。
试验例5：A7R对人脐静脉内皮细胞HUVEC的体外靶向性
取对数生长期的单层培养的人脐静脉内皮细胞(HUVEC细胞)，用0.25％胰蛋白酶消化单层培养细胞，用含10％胎牛血清的DMEM培养液配成单细胞悬液，以每孔1×105个细胞接种于12孔培养板中，每孔体积1mL，将培养板移入二氧化碳培养箱中，37℃、5％CO2及饱和湿度条件下培养24h后，用含10％胎牛血清的DMEM培养液配制浓度为5μM的FITC、DA7R-Fluorescein、cA7R-Fluorescein及LA7R-Fluorescein溶液。将培养板中的培养液吸出，分别加入上述溶液，37℃孵育4h，吸弃上清液。用PBS溶液洗三次，甲醛固定液固定细胞，DAPI进行细胞核染色后，激光共聚焦观察，细胞内化照片见附图17A。另用PBS洗三次后，进行流式细胞仪分析，结果见附图17B。可见HUVEC细胞对DA7R、cA7R和LA7R的摄取明显高于游离荧光素，但对三种多肽的摄取没有明显差异。
试验例6：A7R对U87体外拟态血管模型的体外靶向性
取24孔培养板每孔加入50μL基质胶，平铺于24孔板内，37℃培养箱内孵育30min待其凝固。0.25％胰酶消化U87细胞，用含10％FBS的DMEM 培养液配成单细胞悬液，以每孔1×105个细胞接种于24孔培养板中，37℃、5％CO2及饱和湿度条件下培养12h后血管样结构形成。用含10％胎牛血清的DMEM培养液配制浓度为5μM的FITC、DA7R-Fluorescein、cA7R-Fluorescein及LA7R-Fluorescein溶液。将培养板中的培养液吸出，分别加入上述溶液，37℃孵育4h，吸弃上清液。用PBS溶液洗三次，甲醛固定液固定细胞，DAPI进行细胞核染色后，荧光显微镜观察，照片见附图18。可见U87拟态血管体外模型对DA7R、cA7R和LA7R的摄取明显高于游离荧光素，但对三种多肽的摄取没有明显差异。
试验例7：A7R体内肿瘤靶向性验证
首先构建皮下瘤动物模型。将处于对数生长期的U87细胞胰酶消化，调整细胞浓度为3×107个/mL，接种100μL至裸小鼠右背侧近腋部皮下。接种后饲养于SPF级，定期观察肿瘤大小，待肿瘤大小为200mm3时，筛选出无坏死、肿瘤形状规则的荷瘤裸鼠，分组进行试验。以0.15μmoL/只的剂量将FITC、DA7R-Fluorescein、cA7R-Fluorescein及LA7R-Fluorescein溶液通过尾静脉注入荷瘤裸鼠动物模型体内，1h后处死裸鼠，取出肿瘤，用活体成像仪检测肿瘤的荧光分布，结果见附图19。图A为荷U87皮下移植瘤裸鼠尾静脉注射Fluorescein标记多肽1h后的离体肿瘤影像分布结果；图B为离体脏器的影像分布结果；图C为离体肿瘤的荧光半定量结果；图D为离体肿瘤和脏器的荧光半定量结果。由图可见，Fluorescein标记的DA7R、cA7R和LA7R在肿瘤内的蓄积均显著高于游离荧光素(*p<0.05,**p<0.005)，且肿瘤靶向效果依次为：DA7R>cA7R>LA7R。
试验例8：A7R-Aldoxorubicin对脑胶质瘤细胞U87的体外靶向性
取对数生长期的单层培养的脑胶质瘤细胞(U87细胞)，用0.25％胰蛋白酶消化单层培养细胞，用含10％胎牛血清的DMEM培养液配成单细胞悬液，以每孔1×105个细胞接种于12孔培养板中，每孔体积1mL，将培养板移入二氧化碳培养箱中，37℃、5％CO2及饱和湿度条件下培养24h后，用含10％胎牛血清的DMEM培养液配制浓度为5μM的DOX、Aldoxorubicin、DA7R-Aldoxorubicin及LA7R-Aldoxorubicin溶液。将培养板中的培养液吸出，分别加入上述溶液，37℃孵育4h，吸弃上清液。用PBS溶液洗三次，甲醛固定液固定细胞，DAPI进行细胞核染色后，激光共聚焦观察，细胞内化照片见附图20A。另用PBS洗三次后，进行流式细胞仪分析，结果见附图20B。可见U87细胞对A7R-Aldoxorubicin均有摄取。
试验例9：A7R-Aldoxorubicin对人脐静脉内皮细胞HUVEC的体外靶向性
取对数生长期的单层培养的人脐静脉内皮细胞(HUVEC细胞)，同上试验，细胞内化照片见附图21A。流式细胞仪结果见附图21B。可见HUVEC细胞对A7R-Aldoxorubicin均有摄取。
试验例10：A7R-Aldoxorubicin在不同pH下的稳定性
将DA7R-Aldoxorubicin及LA7R-Aldoxorubicin分别配成1mg/mL的磷酸盐溶液(pH 5.5、6.5、7.4)，37℃孵育，分别于0、0.5、1、2、4、8、12和24h取出20μL溶液，进行HPLC分析。pH稳定性结果表明， LA7R-Aldoxorubicin(附图22A)与DA7R-Aldoxorubicin(附图22B)在pH7.4时最稳定，pH5.5时水解最快，而弱酸性条件(pH6.5)也能很好地降解，说明腙键连接的A7R-Aldoxorubicin在弱酸性环境下能可释放游离阿霉素。
试验例11：A7R-Aldoxorubicin在血清中的稳定性
将DA7R-Aldoxorubicin及LA7R-Aldoxorubicin配成1mg/mL水溶液，取0.1mL加入0.9mL的25％小鼠血清中，37℃孵育，分别于0、0.25，0.5、1、2、4、8和12h取出100μL反应液，加入20μL乙腈沉淀血清中蛋白，4℃静置20min，12000转/分钟离心10min，取上清液20μL进行HPLC分析。由图(附图23)可见，DA7R-Aldoxorubicin在25％小鼠血清中的稳定性显著高于LA7R-Aldoxorubicin，孵育2h，LA7R-Aldoxorubicin完全降解而DA7R-Aldoxorubicin降解不足30％，结果表明，DA7R-Aldoxorubicin具有比LA7R-Aldoxorubicin更好的血清稳定性。。
试验例12：A7R-Aldoxorubicin体内肿瘤靶向性验证
以10mg/Kg的剂量将DA7R-Aldoxorubicin、LA7R-Aldoxorubicin、Aldoxorubicin及DOX通过尾静脉注入荷瘤裸鼠动物模型体内，1h后处死裸鼠，取出肿瘤，用OCT包埋剂(Tissue-Tek)包埋，液氮中快速冷冻，制作10μm的冰冻切片，并在4℃丙酮中固定10min，PBS洗涤，牛血清白蛋白(BSA)孵育封闭1h。将切片与rat anti-mouse CD31(1:10)孵育1h，然后与FITC标记的goat anti-rat IgG(1:100)孵育以定位肿瘤血管，最后切片用DAPI复染以显示细胞核。封片后，用激光共聚焦显微镜观察(附图24)。结果表明，A7R-Aldoxorubicin较Aldoxorubicin和DOX能更好地蓄积在肿瘤组织内，且能与新生血管共定位。DA7R-Aldoxorubicin的靶向效果优于LA7R-Aldoxorubicin。
试验例13：A7R-Aldoxorubicin体外药效试验
以4.0×103个/孔将U87细胞或HUVEC细胞接种于96孔板，24h后，将培养液吸出，加入200μL一系列浓度的DA7R-Aldoxorubicin、LA7R-Aldoxorubicin、Aldoxorubicin及DOX，培养72h后加入MTT溶液继续培养4h，弃去培养液，加入150μL DMSO，振荡至紫色颗粒溶解，用酶标仪在590nm处测定吸光度值。采用MTT法测定细胞存活率，计算细胞存活率和半数致死剂量(附图25)。图A和图B分别为DA7R-Aldoxorubicin、LA7R-Aldoxorubicin、Aldoxorubicin和DOX抗U87细胞和HUVEC细胞的活性曲线。图A表明U87细胞给药培养72h后，其IC50分别为6.76、13.18、2.95和0.081μM。四种化合物均具能抑制U87细胞的体外生长。图B表明HUVEC细胞给药培养72h后，其IC50分别为0.63、1.047、0.891和0.0105μM。四种化合物均具能抑制HUVEC细胞的体外生长。
试验例14：A7R-Aldoxorubicin对皮下移植瘤抑制试验
构建U87皮下瘤动物模型，定期观察肿瘤大小，待肿瘤大小为100mm3时，分组进行试验，分别尾静脉注射DA7R-Aldoxorubicin、LA7R-Aldoxorubicin、Aldoxorubicin、DOX以及生理盐水。给药组阿霉素总给药剂量为2.5mg/kg，分为五次，每次给药间隔为两天。隔天以游标卡尺测量肿瘤的长径(a)及短径(b)。根据公式计算各组裸鼠肿瘤体积，绘制肿 瘤体积随时间的变化曲线，计算各组统计学差异。按照以下公式计算肿瘤体积：
V瘤体积＝0.5(a×b2)
给药14天后，断颈处死所有裸鼠，取下皮下肿瘤并称重，计算各组统计学差异(附图26)。图A为各组裸鼠肿瘤体积随时间变化的曲线，图B为各组裸鼠体重随时间变化的曲线，图C为将裸鼠处死取出肿瘤组织后称重的统计分析结果，由图可见A7R-Aldoxorubicin的抗肿瘤效果优于未修饰的DOX，其中DA7R-Aldoxorubicin的药效最佳。
试验例15：不同剂量A7R-Aldoxorubicin对皮下移植瘤抑制试验
给药组高、中、低三剂量，按阿霉素总给药剂量分别为2.5mg/kg、7.5mg/kg、22.5mg/kg，多肽DA7R剂量为12mg/kg，同上实验。结果见附图27。图A为各组裸鼠肿瘤体积随时间变化的曲线，图B为各组裸鼠体重随时间变化的曲线，图C为将裸鼠处死取出肿瘤组织后称重的统计分析结果，由图可见，给药剂量为2.5mg/kg和7.5mg/kg时DA7R-Aldoxorubicin的抗肿瘤效果优于DOX，给药剂量为22.5mg/kg时，DOX组裸鼠体重下降20％，而DA7R-Aldoxorubicin组体重没有明显变化(n＝8，***p<0.001，*p<0.05)。
试验例16：A7R-Aldoxorubicin抑制肿瘤血管及促凋亡作用
DA7R-Aldoxorubicin、LA7R-Aldoxorubicin、Aldoxorubicin、DOX组经过五次尾静脉注射后，处死取出皮下瘤固定，石蜡包埋切片。进行CD31免疫组化染色与PAS双染考察血管抑制效果。采用末端脱氧核苷酸转移酶(TDT)介导的dUTP缺口末端标记法(Terminal deoxynucleotidyl Transferase-mediated dUTP nick end labeling，TUNEL)检测肿瘤细胞的凋亡程度。主要步骤为：石蜡切片常规脱蜡至水；PBS漂洗3次，每次3min；0.3％H2O2溶液室温处理20min；20μg/mL蛋白酶K 37℃消化20min；PBS漂洗3次，每次3min；每张切片滴加TUNEL混合液(TDT和biotin-dNTP)30μL置于湿盒中37℃孵育60min；PBS漂洗3min共3次；Streptavidin-HRP(1:200)37℃孵育30min；PBS漂洗3次，每次3min；0.04％DAB+0.03％H2O2溶液显色10min，水洗；苏木素衬染1min，水洗蓝化；吹干后常规树脂封片。结果见附图28。附图28中，第一排为DA7R-Aldoxorubicin、LA7R-Aldoxorubicin、Aldoxorubicin和DOX抑制新生血管形成的CD31/PAS双染色照片(bar＝100μm)，其中新生血管细胞核呈棕黄色或棕褐色。与LA7R-Aldoxorubicin相比，DA7R-Aldoxorubicin抑制新生血管的形成更显著。第二排为DA7R-Aldoxorubicin、LA7R-Aldoxorubicin、Aldoxorubicin和DOX促进皮下瘤凋亡的TUNEL染色照片(bar＝100μm)，其中凋亡的阳性细胞核呈棕黄色或棕褐色。与LA7R-Aldoxorubicin相比，DA7R-Aldoxorubicin促进肿瘤组织的凋亡更显著。
试验例17：不同剂量A7R-Aldoxorubicin抑制肿瘤血管及促凋亡作用
高、中、低三剂量DA7R-Aldoxorubicin、DOX组及DA7R组经过五次尾静脉注射后，同上试验。结果见附图29，其中，图A为不同剂量DA7R-Aldoxorubicin和DOX及多肽DA7R抑制新生血管形成的CD31/PAS双染色照片(bar＝100μm)，其中新生血管细胞呈棕黄色或棕褐色。相同剂 量下，与DOX相比，DA7R-Aldoxorubicin抑制新生血管的形成更显著，图B为不同剂量DA7R-Aldoxorubicin和DOX及多肽DA7R促进皮下瘤凋亡的TUNEL染色照片(bar＝100μm)，其中凋亡的阳性细胞核呈棕黄色或棕褐色。相同剂量下，DA7R-Aldoxorubicin比DOX促进肿瘤组织的凋亡更显著。
试验例18：A7R-Aldoxorubicin的全身毒性考察
将皮下瘤药效学试验的各给药组小鼠的心、肝、脾、肺、肾组织解剖后固定于4％多聚甲醛的PBS溶液中，石蜡包埋切片，进行HE染色，在显微镜下观察、拍摄图片(附图30)。图为皮下瘤药效实验中各组裸鼠的心、肝、脾、肺、肾HE染色结果，表明药效实验中各组药物对裸鼠脏器没有明显毒性。
试验例19：A7R-PEG-脂质体体外细胞靶向性验证
取对数生长期的单层培养的脑胶质瘤细胞(U87细胞)，用0.25％胰蛋白酶消化单层培养细胞，用含10％胎牛血清的DMEM培养液配成单细胞悬液，以每孔1×105个细胞接种于12孔培养板中，每孔体积1mL，将培养板移入二氧化碳培养箱中，37℃、5％CO2及饱和湿度条件下培养24h后，用含10％胎牛血清的DMEM培养液配制浓度为5μM的mPEG-脂质体/FAM、DA7R-PEG-脂质体/FAM、cA7R-PEG-脂质体/FAM和LA7R-PEG-脂质体/FAM溶液。将培养板中的培养液吸出，分别加入上述溶液，37℃孵育4h，吸弃上清液。用PBS溶液洗三次，甲醛固定液固定细胞，DAPI进行细胞核染色后，激光共聚焦观察，细胞内化照片见附图31A。另用PBS洗三次后，进行流式细胞仪分析，结果见附图31B。可见U87细胞对DA7R、cA7R和LA7R修饰脂质体的摄取量显著高于无多肽修饰脂质体。
试验例20：A7R-PEG-脂质体对人脐静脉内皮细胞HUVEC的体外靶向性
取对数生长期的单层培养的人脐静脉内皮细胞(HUVEC细胞)，同上试验，细胞内化照片见附图32A，流式细胞仪结果见附图32B。由图可知，HUVEC细胞对多肽修饰脂质体的摄取明显高于无多肽修饰脂质体。
试验例21：A7R-PEG-脂质体对U87体外拟态血管模型的体外靶向性
取24孔培养板每孔加入50μL基质胶，平铺于24孔板内，37℃培养箱内孵育30min待其凝固。0.25％胰酶消化U87细胞，用含10％FBS的DMEM培养液配成单细胞悬液，以每孔1×105个细胞接种于24孔培养板中，37℃，5％CO2及饱和湿度条件下培养12h后血管样结构形成。用含10％胎牛血清的DMEM培养液配制浓度为5μM的mPEG-脂质体/FAM、DA7R-PEG-脂质体/FAM、cA7R-PEG-脂质体/FAM和LA7R-PEG-脂质体/FAM溶液。将培养板中的培养液吸出，分别加入上述溶液，37℃孵育4h，吸弃上清液。用PBS溶液洗三次，甲醛固定液固定细胞，DAPI进行细胞核染色后，荧光显微镜观察，照片见附图33。由图可知，U87拟态血管体外模型对多肽修饰脂质体的摄取明显高于无多肽修饰脂质体。
试验例22：A7R-PEG-脂质体体内靶向性验证
U87皮下瘤模型裸鼠分别尾静脉注射100μL包载DiR的脂质体mPEG- 脂质体/DiR、DA7R-PEG-脂质体/DiR、cA7R-PEG-脂质体/DiR和LA7R-PEG-脂质体/DiR溶液，分别在注射后2、4、8、12及24h时麻醉小鼠，用活体成像仪记录DiR荧光在裸鼠体内的分布情况并进行荧光半定量计算(附图34)。图A为尾静脉注射24小时后的在体荧光分布图像。图B为给药后各个时间点肿瘤内荧光强度半定量结果。图C为脏器的荧光分布图像。图D为图C的荧光半定量统计结果。结果表明DA7R或cA7R修饰的脂质体能更好地靶向至肿瘤部位。
试验例23：载阿霉素的A7R-PEG-脂质体体外药效学试验
以4.0×103个/孔将U87细胞或HUVEC细胞接种于96孔板，24h后，将培养液吸出，加入200μL一系列浓度的mPEG-脂质体/DOX、DA7R-PEG-脂质体/DOX、cA7R-PEG-脂质体/DOX、LA7R-PEG-脂质体/DOX及游离DOX，共培养4h后吸出药液，PBS洗涤后加入含10％FBS的DMEM培养液，继续培养68h，后加入MTT溶液继续培养4h，弃去培养液，加入150μL DMSO，振荡至紫色颗粒溶解，用酶标仪在590nm处测定吸光度值。采用MTT法测定细胞存活率，计算细胞存活率和半数致死剂量(附图35)。图A和图B分别为LS/DOX、DA7R-LS/DOX、cA7R-LS/DOX、LA7R-LS/DOX和DOX抗U87细胞和HUVEC细胞的活性曲线。图A表明U87细胞给药4h培养72h后，其IC50分别为17.78、0.62、0.81、5.13和0.06μM。四种脂质体均具能抑制U87细胞的体外生长，其中DA7R-LS/DOX和cA7R-LS/DOX的体外活性分别为LA7R-LS/DOX的8.27倍和6.33倍。图B表明HUVEC细胞给药4h培养72h后，其IC50分别为0.71、0.19、0.15、0.39和0.09μM。四种脂质体均具能抑制HUVEC细胞的体外生长，其中DA7R-LS/DOX和cA7R-LS/DOX的体外活性分别为LA7R-LS/DOX的2.05倍和2.60倍。
试验例24：载阿霉素的A7R-PEG-脂质体对新生血管形成的抑制试验
取24孔培养板每孔加入50μL基质胶，平铺于24孔板内，37℃培养箱内孵育30min待其凝固。0.25％胰酶消化HUVEC细胞，用含1μM阿霉素脂质体或游离阿霉素药液的DMEM培养液配成单细胞悬液，以每孔1×105个细胞接种于24孔培养板中，37℃、5％CO2及饱和湿度条件下培养12h后观察血管样结构形成(附图36A)并计算血管样结构的形成率(附图36B)。结果表明，相比于LA7R-LS/DOX，DA7R-LS/DOX和cA7R-LS/DOX能显著抑制新生血管的形成(**p<0.005)。
试验例25：载阿霉素的A7R-PEG-脂质体对拟态血管形成的抑制试验
取24孔培养板每孔加入50μL基质胶，平铺于24孔板内，37℃培养箱内孵育30min待其凝固。0.25％胰酶消化U87细胞，用含1μM阿霉素脂质体或游离阿霉素药液的DMEM培养液配成单细胞悬液，以每孔1×105个细胞接种于24孔培养板中，37℃、5％CO2及饱和湿度条件下培养12h后观察血管样结构形成(附图37A)并计算拟态血管结构的形成率(附图37B)。结果表明，相比于LA7R-LS/DOX，DA7R-LS/DOX和cA7R-LS/DOX能显著抑制拟态血管的形成(*p<0.05)。
试验例26：载阿霉素的A7R-PEG-脂质体对皮下移植瘤抑制试验
构建U87皮下瘤动物模型，定期观察肿瘤大小，待肿瘤大小为100mm3时，分组进行试验，分别尾静脉注射mPEG-脂质体/DOX、DA7R-PEG-脂质体/DOX、cA7R-PEG-脂质体/DOX、LA7R-PEG-脂质体/DOX、游离DOX以及生理盐水。给药组阿霉素总给药剂量为10mg/kg，分为五次，每次给药间隔为两天。隔天以游标卡尺测量肿瘤的长径(a)及短径(b)。根据公式计算各组裸鼠肿瘤体积，绘制肿瘤体积随时间的变化曲线，计算各组统计学差异。按照以下公式计算肿瘤体积：
V瘤体积＝0.5(a×b2)
给药18天后(接种后24天)，断颈处死所有裸鼠，取下皮下肿瘤并称重，计算各组统计学差异(附图38)。图A为各组裸鼠肿瘤体积随时间变化的曲线，与生理盐水组相比，各给药组对肿瘤生长均有抑制作用。DA7R-LS/DOX、cA7R-LS/DOX与LA7R-LS/DOX相比具有极显著性差异(n＝6，***p<0.001)。将裸鼠处死取出肿瘤组织后称重并进行统计分析(图B)，发现DA7R-LS/DOX和cA7R-LS/DOX组瘤重显著低于LA7R-LS/DOX(n＝6，***p<0.001)。
试验例27：载阿霉素的A7R-PEG-脂质体促凋亡试验
不同阿霉素脂质体组及游离阿霉素治疗组经过五次尾静脉注射后，处死取出皮下瘤固定，石蜡包埋切片。采用末端脱氧核苷酸转移酶(TDT)介导的dUTP缺口末端标记法(Terminal deoxynucleotidyl Transferase-mediated dUTP nick end labeling，TUNEL)检测肿瘤细胞的凋亡程度。主要步骤为：石蜡切片常规脱蜡至水；PBS漂洗3次，每次3min；0.3％H2O2溶液室温处理20min；20μg/mL蛋白酶K 37℃消化20min；PBS漂洗3次，每次3min；每张切片滴加TUNEL混合液(TDT和biotin-dNTP)30μL置于湿盒中37℃孵育60min；PBS漂洗3min共3次；Streptavidin-HRP(1:200)37℃孵育30min；PBS漂洗3次，每次3min；0.04％DAB+0.03％H2O2溶液显色10min，水洗；苏木素衬染1min，水洗蓝化；吹干后常规树脂封片。阳性结果为细胞核呈棕黄色或棕褐色。细胞核内棕色颗粒阳性即判定为凋亡细胞。在普通光学显微镜下连续观察5个高倍视野计数阳性细胞数，视野内细胞中阳性细胞数所占的百分比为凋亡指数。结果见附图39。图A为LS/DOX、DA7R-LS/DOX、cA7R-LS/DOX、LA7R-LS/DOX和DOX促进皮下瘤凋亡的TUNEL染色照片(bar＝50μm)。图B为阳性细胞数的统计结果，与LA7R-LS/DOX相比，DA7R-LS/DOX和cA7R-LS/DOX能显著促进肿瘤组织的凋亡。
试验例28：载阿霉素的A7R-PEG-脂质体对肿瘤血管抑制试验
不同阿霉素脂质体组及游离阿霉素治疗组经过五次尾静脉注射后，处死取出皮下瘤固定，石蜡包埋切片，进行CD31免疫组化染色与PAS双染。在普通光学显微镜下连续观察3个高倍视野计数CD31阳性血管数。结果见附图40。图A为LS/DOX、DA7R-LS/DOX、cA7R-LS/DOX、LA7R-LS/DOX和DOX抑制新生血管形成的CD31/PAS双染色照片(bar＝100μm)，其中新生血管细胞核呈棕黄色或棕褐色，图B为新生血管数的统计结果，与LA7R-LS/DOX相比，DA7R-LS/DOX和cA7R-LS/DOX能显著抑制新生血管的形成。
试验例29：DA7R多肽及DA7R多肽与阿霉素联合给药抗U87皮下瘤药效学 试验
构建U87皮下瘤动物模型，定期观察肿瘤大小，待肿瘤大小为100mm3时，分组进行试验，分别尾静脉注射DA7R、LA7R、DA7R与DOX、LA7R与DOX、DOX以及生理盐水。给药组阿霉素总给药剂量为2.5mg/kg，多肽总给药剂量为20mg/kg，分为五次，每次给药间隔为两天。隔天以游标卡尺测量肿瘤的长径(a)及短径(b)。根据公式计算各组裸鼠肿瘤体积，绘制肿瘤体积随时间的变化曲线，计算各组统计学差异。按照以下公式计算肿瘤体积：
V瘤体积＝0.5(a×b2)
给药14天后，断颈处死所有裸鼠，取下皮下肿瘤并称重，计算各组统计学差异(附图41)。图A为各组裸鼠肿瘤体积随时间变化的曲线，图B为各组裸鼠体重随时间变化的曲线，图C为将裸鼠处死取出肿瘤组织后称重的统计分析结果，由图可见，A7R多肽本身具有抗肿瘤活性，且DA7R活性显著优于LA7R。A7R与DOX联合给药显著增强DOX抗肿瘤药效，且DA7R与DOX联合给药比LA7R与DOX联合给药更优(n＝8，***p<0.001，**p<0.01)。
试验例30：DA7R多肽及DA7R多肽与阿霉素联合给药抗U87原位瘤药效学 试验
构建U87原位瘤模型，将原位脑胶质瘤模型鼠随机分成5组(n＝8)，分别在第7，10,13，16,19天尾静脉注射DA7R(单次剂量4mg/kg)，游离阿霉素(DOX，单次剂量2mg/kg)，DA7R与DOX联合给药，DA7R-Aldoxo(按DOX单次剂量2mg/kg)，记录模型裸鼠的生存时间如附图42所示。结果显示，对照组中位生存期为20.5天。DA7R组为23.5天，表明单用DA7R多肽能延长中位生存期。DOX组和DA7R-Aldoxo组中位生存期分别为22.5天和25.5天，有一定的治疗效果。DA7R与DOX联合给药组中位生存期延长至27天，体现了联合用药的优势。
试验例31：DA7R多肽与阿霉素脂质体联合给药抗U87原位瘤药效学试验
构建U87原位瘤模型，将原位脑胶质瘤模型鼠随机分成4组(n＝8)，分别在第7，10,13，16,19天尾静脉注射DA7R(单次剂量4mg/kg)，阿霉素脂质体(LS/DOX，单次剂量2mg/kg)，DA7R与LS/DOX联合给药，记录模型裸鼠的生存时间如附图43所示。结果显示，对照组中位生存期为20.5天。DA7R组为23.5天，表明单用DA7R多肽能延长中位生存期。LS/DOX组中位生存期为26天，有一定的治疗效果。DA7R与LS/DOX联合给药组中位生存期延长至31.5天，体现了联合用药的优势。
试验例32：DA7R与替莫唑胺(TMZ)联合用药的药效学试验
构建U87原位瘤模型，将原位脑胶质瘤模型鼠随机分成4组(n＝10)，分别在第10，12,14，16,18，20,22天尾静脉注射DA7R(单次剂量4mg/kg)或者灌胃TMZ(单次剂量10mg/kg)，记录模型裸鼠的生存时间，结果见附 图44。结果显示，对照组中位生存期为25天。DA7R组为26天，表明单用DA7R多肽能延长中位生存期。TMZ组中位生存期为43天，表现出较好的治疗效果。DA7R与TMZ联合给药组中位生存期延长至50天，体现了联合用药的优势。
试验例33：DA7R多肽与阿霉素联合给药抗A549皮下瘤药效学试验
构建A549皮下瘤模型，将模型鼠随机分成6组(n＝6)，分别尾静脉注射DA7R(总剂量20mg/kg)，游离阿霉素(DOX，总剂量5mg/kg)，阿霉素脂质体(LS/DOX，总剂量5mg/kg)，DA7R与DOX联合给药，DA7R与LS/DOX联合给药，隔天监测裸鼠瘤体积，并在最后一天处死裸鼠称取瘤重。
按照以下公式计算肿瘤体积：
V瘤体积＝0.5(a×b2)
结果见附图45。由图可见，DA7R本身具有抗A549皮下瘤的作用，DA7R与DOX或LS/DOX联合给药均显著增强抗肿瘤药效。
试验例34：A7R与DOX联合给药体内肿瘤靶向性验证
以阿霉素10mg/kg，多肽10mg/kg的剂量将DA7R与DOX联合给药、LA7R与DOX联合给药及DOX通过尾静脉注入荷瘤裸鼠动物模型体内，1h后处死裸鼠，取出肿瘤，用O.C.T.包埋剂(Tissue-Tek)包埋，液氮中快速冷冻，制作10μm的冰冻切片，并在4℃丙酮中固定10min，PBS洗涤，牛血清白蛋白(BSA)孵育封闭1h。将切片与rat anti-mouse CD31(1:10)孵育1h，然后与FITC标记的goat anti-rat IgG(1:100)孵育以定位肿瘤血管，最后切片用DAPI复染以显示细胞核。封片后，用激光共聚焦显微镜观察(附图46)。结果表明，较LA7R与DOX联合给药或DOX给药，DA7R与DOX联合给药能更好地使DOX蓄积在肿瘤组织内，且能与新生血管共定位。说明DA7R与DOX联合给药可增加皮下瘤内DOX的蓄积量。
试验例35：最大耐受剂量(MTD)试验
昆明种雄性小鼠，一组3只，分别给药DOX(5mg/kg；10mg/kg；15mg/kg)；Aldoxorubicin(按DOX剂量5mg/kg；10mg/kg；15mg/kg)；
DA7R-Aldoxorubicin(按DOX剂量5mg/kg；10mg/kg；15mg/kg)；多肽DA7R(10mg/kg；20mg/kg；30mg/kg；40mg/kg；50mg/kg；60mg/kg)；DA7R与DOX联合给药(DA7R 8.67mg/kg+DOX 5mg/kg；DA7R 17.35mg/kg+DOX10mg/kg；DA7R 26.02mg/kg+DOX 15mg/kg)。每天记录小鼠体重变化，连续记录8天，观察有无小鼠死亡。MTD为小鼠没有死亡且体重减轻<15％时的剂量。
相对体重变化＝每天体重/初始体重×100％
结果见附图47。图A为不同剂量下DOX组小鼠体重变化曲线，图B为不同剂量下Aldoxorubicin组小鼠体重变化曲线，图C为不同剂量下DA7R组小鼠体重变化曲线，图D为不同剂量下DA7R与DOX联合给药组小鼠体重变化曲线，图E为不同剂量下DA7R-Aldoxorubicin组小鼠体重变化曲线，图F为第8天各组体重变化条形统计图。由图可知，多肽DA7R的MTD大于60mg/kg，DOX的MTD约为10mg/kg，DA7R-Aldoxorubicin的MTD约为 15mg/kg。
试剂盒
本发明还提供了一种试剂盒，该试剂盒包括本发明提供的稳定化A7R多肽，稳定化A7R多肽复合物或递药系统。
此外，所述试剂盒中还可包括使用说明书。
药物组合物
本发明还涉及一种用于诊断、示踪和/或治疗肿瘤的药物组合物，其包含药学上可接受的载体，以及本发明提供的稳定化A7R多肽，稳定化A7R多肽复合物或递药系统。所述提供的稳定化A7R多肽，稳定化A7R多肽复合物或递药系统在该药物组合物中可以为有效量的或治疗有效量的。
如本文所用，“有效量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。
如本文所用，“药学上可接受的”成分是适用于人和/或动物(如哺乳动物或禽类)而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。“药学上可接受的载体”是指用于给药的载体，可以包括各种赋形剂和稀释剂等。这类载体可以包括但不限于：水、生理盐水、脂质体、脂质、蛋白、蛋白-抗体缀合物、肽类物质、纤维素、纳米凝胶、缓冲液、葡萄糖、甘油、乙醇及其组合。载体的选择通常应与给药方式相匹配，这是本领域的普通技术人员所熟知的。
本发明所述的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的活性成分的药代动力学参数，例如生物利用度、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。
诊断/示踪方法
本发明还提供了一种诊断/示踪肿瘤的方法，该方法包括使用本发明提供的稳定化A7R多肽，稳定化A7R多肽复合物或递药系统。
治疗方法
本发明还提供了一种治疗肿瘤的方法，所述方法包括向受试者给药治疗有效量的本发明提供的稳定化A7R多肽，稳定化A7R多肽复合物或递药系统。
所述口服或非口服可以通过胃肠道、鼻腔、气管、肺、非病灶部位的静脉或表皮、皮内、皮下、心内、肌肉、骨髓、腹腔、硬膜外、口腔、舌下、眼部、直肠、阴道、尿道、耳道等途径给药。优选施用方式或给药方式包括：口服、呼吸道、注射、透皮、粘膜、或腔道给药。
其中，所述口服给药方式包括吞服、含化等。所述呼吸道给药方式包括吸入方式，例如超声雾化吸入、氧气雾化吸入、手压式雾化吸入等。所述注射给药方式包括动脉注射、静脉注射、肌肉注射、心内注射、皮内注射等。 所述透皮给药或经皮给药方式，包括离子导入法、电致孔透皮法等。所述粘膜给药方式包括鼻粘膜给药、口腔粘膜给药、眼粘膜给药、直肠粘膜给药、子宫给药以及阴道粘膜给药等。所述腔道给药方式包括直肠给药、阴道给药、尿道给药、鼻腔给药、耳道给药等。
在本发明提及的所有文献(包括专利文献或非专利文献)都在本发明中引用作为参考，就如同每一篇文献被单独引用作为参考那样。
尽管本发明已进行了一定程度的描述，明显地，在不脱离本发明的精神和范围的条件下，可进行各个条件的适当变化。可以理解，本发明不限于所述实施方案，而归于权利要求的范围，其包括所述每个因素的等同替换。
Figure PCTCN2016101738-appb-000001

Claims (15)

  1. 一种稳定化A7R多肽，其特征在于，所述A7R多肽为逆序D构型多肽DA7R和/或首尾酰胺键环合多肽cA7R，其中，所述逆序D构型多肽DA7R的氨基酸序列为DRDPDPDLDWDTDA，所述首尾酰胺键环合多肽cA7R的L构型氨基酸序列为c(CATWLPPR)。
  2. 一种稳定化A7R多肽复合物，其特征在于，所述稳定化A7R多肽复合物为权利要求1所述的稳定化A7R多肽修饰含有马来酰亚胺基团的影像物质，其中，所述稳定化A7R多肽复合物的结构为DA7R-X和/或cA7R-X，X为所述影像物质；
    优选地，所述X选自荧光物质、近红外染料和磁共振影像剂中的一种或多种；
    更优选地，所述荧光物质为5-羧基荧光素，所述近红外染料选自cy5.5、IR820和DiR中的一种或多种，所述磁共振影像剂为Gd-DTPA。
  3. 一种权利要求2所述稳定化A7R多肽复合物的制备方法，其特征在于，所述方法包括：权利要求1所述的稳定化A7R多肽或巯基化的权利要求1所述的稳定化A7R多肽与含有马来酰亚胺基团的影像物质反应。
  4. 一种稳定化A7R多肽复合物，其特征在于，所述稳定化A7R多肽复合物为权利要求1所述的稳定化A7R多肽修饰抗肿瘤药物，其中，所述稳定化A7R多肽复合物的结构为DA7R-Y和/或cA7R-Y，Y为所述抗肿瘤药物；
    优选地，所述抗肿瘤药物选自含酮或醛基的药物、含羟基或氨基的药物、含硼酸基团的药物和多肽药物中的一种或多种；
    更优选地：
    所述含酮或醛基的药物为阿霉素或表阿霉素，
    所述含羟基或氨基的药物选自紫杉醇、多烯紫杉醇、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、依托泊苷、吉西他滨、阿糖胞苷、5-氟尿嘧啶、替尼泊苷、莫立替尼、埃博霉素、长春瑞滨、放线菌素D、米托蒽醌、丝裂霉素、博来霉素、伊立替康，
    所述含硼酸基团的药物为硼替佐米或卡非佐米，和/或
    所述多肽药物选自p53激活肽、蜂毒肽和蝎毒肽中的一种或多种。
  5. 一种权利要求4所述稳定化A7R多肽复合物的制备方法，其特征在于，所述方法包括：
    当所述抗肿瘤药物为含酮或醛基的药物时，权利要求1所述的稳定化A7R多肽通过pH敏感腙键与所述抗肿瘤药物连接制备所述稳定化A7R多肽复合物；
    当所述抗肿瘤药物为含羟基或氨基的药物时，权利要求1所述的稳定化A7R多肽通过二硫键与所述抗肿瘤药物连接制备所述稳定化A7R多肽复合物；
    当所述抗肿瘤药物为含硼酸基团的药物时，权利要求1所述的稳定化 A7R多肽通过pH敏感的硼酸脂键与所述抗肿瘤药物连接制备所述稳定化A7R多肽复合物；和/或
    当所述抗肿瘤药物为多肽药物时，权利要求1所述的稳定化A7R多肽与所述抗肿瘤药物缩合制备所述稳定化A7R多肽复合物。
  6. 一种稳定化A7R多肽复合物，其特征在于，所述稳定化A7R多肽复合物为权利要求1所述的稳定化A7R多肽修饰高分子载体材料，其中，所述稳定化A7R多肽复合物的结构为DA7R-聚乙二醇-Z和/或cA7R-聚乙二醇-Z，Z为所述高分子载体材料；
    优选地，所述高分子载体材料选自磷脂、聚乳酸、乳酸羟基乙酸共聚物和聚己内酯中的一种或多种。
  7. 一种权利要求6所述稳定化A7R多肽复合物的制备方法，其特征在于，所述方法包括：
    通过权利要求1所述的稳定化A7R多肽或巯基化的权利要求1所述的稳定化A7R多肽与马来酰亚胺-聚乙二醇-高分子载体材料反应，制备所述稳定化A7R多肽复合物。
  8. 一种递药系统，其特征在于，所述递药系统包括权利要求6所述的稳定化A7R多肽复合物；优选地，所述递药系统为脂质体递药系统、聚合物胶束递药系统、聚合物圆盘递药系统或纳米粒递药系统。
  9. 根据权利要求8所述的递药系统，其特征在于，所述递药系统还包括所述稳定化A7R多肽复合物以外的(1)影像物质和/或(2)抗肿瘤药物；优选地：
    所述(1)影像物质选自荧光物质、近红外染料和磁共振影像剂中的一种或多种，更优选地，所述荧光物质为5-羧基荧光素，所述近红外染料选自Cy5.5、IR820、DiR和DiD中的一种或多种，和/或所述磁共振影像剂为Gd-DTPA，和/或
    所述(2)抗肿瘤药物选自：阿霉素、表阿霉素、紫杉醇、多烯紫杉醇、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、硼替佐米、卡非佐米、环磷酰胺、依托泊苷、吉西他滨、阿糖胞苷、5-氟尿嘧啶、替尼泊苷、莫立替尼、埃博霉素、长春瑞滨、放线菌素D、米托蒽醌、丝裂霉素、博来霉素、伊立替康、顺铂、奥沙利铂、p53激活肽、蜂毒肽、蝎毒肽、贝伐单抗和曲妥单抗中的一种或多种。
  10. 权利要求1所述的稳定化A7R多肽、权利要求2、4、6中任一项所述的稳定化A7R多肽复合物、权利要求8或9所述的递药系统在制备用于诊断、示踪和/或治疗肿瘤的药品或医疗产品中的应用，优选地：
    所述肿瘤为高表达新生血管内皮生长因子受体2肿瘤或高表达神经纤毛蛋白-1肿瘤。
  11. 一种用于诊断、示踪和/或治疗肿瘤的组合产品，其特征在于，所述组合产品包括选自以下的一种或多种成分：权利要求1所述的稳定化A7R多 肽、权利要求2、4、6中任一项所述的稳定化A7R多肽复合物、权利要求8或9所述的递药系统；优选地：
    所述组合产品为试剂盒，和/或
    所述肿瘤为高表达新生血管内皮生长因子受体2肿瘤或高表达神经纤毛蛋白-1肿瘤。
  12. 一种诊断、示踪和/或治疗肿瘤的方法，其特征在于，所述方法包括对患有所述肿瘤或疑患有所述肿瘤的患者通过口服或非口服途径给予有效剂量的选自以下的一种或多种物质：
    权利要求1所述的稳定化A7R多肽；
    权利要求2、4、6中任一项所述的稳定化A7R多肽复合物；
    权利要求8或9所述的递药系统；
    权利要求11所述的组合产品；
    优选地，所述肿瘤为高表达新生血管内皮生长因子受体2肿瘤或高表达神经纤毛蛋白-1肿瘤；和/或
    优选地，当所述方法用于治疗肿瘤时，所述方法包括对患有所述肿瘤或疑患有所述肿瘤的患者通过口服或非口服途径给予有效剂量的(1)权利要求1所述的稳定化A7R多肽与(2)一种或多种其他抗肿瘤药物，所述其他抗肿瘤药物优选选自：阿霉素、表阿霉素、紫杉醇、多烯紫杉醇、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、硼替佐米、卡非佐米、环磷酰胺、替莫唑胺、依托泊苷、巯嘌呤、吉西他滨、阿糖胞苷、5-氟尿嘧啶、替尼泊苷、莫立替尼、埃博霉素、长春瑞滨、放线菌素D、米托蒽醌、丝裂霉素、博来霉素、伊立替康、顺铂、奥沙利铂、p53激活肽、蜂毒肽、蝎毒肽、贝伐单抗和曲妥单抗。
  13. 权利要求1所述的稳定化A7R多肽作为抗肿瘤活性成分在制备用于抗肿瘤的药品和/或医疗产品中的应用。
  14. 权利要求1所述的稳定化A7R多肽在制备肿瘤靶向产品中的应用，优选地：
    所述肿瘤靶向产品用于靶向VEGFR2和NRP-1高表达的肿瘤新生血管、拟态血管和肿瘤细胞；和/或
    所述肿瘤靶向产品为用于诊断、示踪和/或治疗肿瘤的药品、实验试剂和/或医疗产品。
  15. 权利要求1所述的稳定化A7R多肽在制备用于协同增效其他抗肿瘤药物疗效的药品、实验试剂和/或医疗产品中的应用；
    优选地，所述其他抗肿瘤药物优选选自：阿霉素、表阿霉素、紫杉醇、多烯紫杉醇、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、硼替佐米、卡非佐米、环磷酰胺、替莫唑胺、依托泊苷、巯嘌呤、吉西他滨、阿糖胞苷、5-氟尿嘧啶、替尼泊苷、莫立替尼、埃博霉素、长春瑞滨、放线菌素D、米托蒽醌、丝裂霉素、博来霉素、伊立替康、顺铂、奥沙利铂、p53激活肽、蜂毒肽、蝎毒肽、贝伐单抗和曲妥单抗中的一种或多种。
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CN110386962A (zh) * 2019-07-04 2019-10-29 苏州强耀生物科技有限公司 一种阿霉素偶联靶向多肽的合成方法
CN112279887A (zh) * 2020-10-29 2021-01-29 首都医科大学 氨基酸衍生物及其制备方法和应用、一种抗肿瘤胶束及其制备方法
CN113372412A (zh) * 2021-04-09 2021-09-10 上海交通大学医学院附属第九人民医院 一种治疗骨肿瘤的细胞靶向多肽及其制备方法和用途
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CN113113143B (zh) * 2021-04-12 2024-03-01 重庆大学 一种考虑延迟强化核磁影像的心肌梗死危险程度评估系统
CN113337468A (zh) * 2021-05-27 2021-09-03 上海市伤骨科研究所 一种双功能化外泌体及其制备方法和在脑卒中修复的应用
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CN113384682B (zh) * 2021-05-31 2023-07-04 南方医科大学 蝎毒多肽Smp43在制备抗肿瘤药物的应用

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