WO2019237884A1 - 一种淀粉样蛋白β短肽介导的脑靶向递送系统及其制备方法和用途 - Google Patents

一种淀粉样蛋白β短肽介导的脑靶向递送系统及其制备方法和用途 Download PDF

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WO2019237884A1
WO2019237884A1 PCT/CN2019/087363 CN2019087363W WO2019237884A1 WO 2019237884 A1 WO2019237884 A1 WO 2019237884A1 CN 2019087363 W CN2019087363 W CN 2019087363W WO 2019237884 A1 WO2019237884 A1 WO 2019237884A1
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delivery system
brain
nano
drug
polyethylene glycol
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French (fr)
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占昌友
张醉
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Fudan University
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Fudan University
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Priority to CN201980032453.3A priority Critical patent/CN112203693B/zh
Priority to JP2020567491A priority patent/JP7628699B2/ja
Priority to US17/251,061 priority patent/US12178885B2/en
Priority to EP19819733.7A priority patent/EP3834844A4/en
Publication of WO2019237884A1 publication Critical patent/WO2019237884A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/65—Tetracyclines
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6925—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a microcapsule, nanocapsule, microbubble or nanobubble
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0089—Particulate, powder, adsorbate, bead, sphere
    • A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
    • A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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

Definitions

  • the invention belongs to the field of pharmacy, and relates to a brain-targeted delivery system mediated by amyloid beta short peptide (SP), in particular to a specific apolipoprotein that can be adsorbed in plasma and can mediate drugs across the blood-brain barrier
  • SP amyloid beta short peptide
  • the inventors of the present application intend to provide a brain-targeted delivery system mediated by an amyloid beta short peptide (hereinafter abbreviated as SP), and more particularly, it relates to a plasma apolipoprotein that can be specifically adsorbed Polypeptide modified complexes and targeted delivery systems capable of mediating drugs across the blood-brain barrier and their use in the preparation of preparations for the diagnosis and treatment of brain tumors (including brain metastases and primary brain tumors) and other brain diseases Applications.
  • SP amyloid beta short peptide
  • the peptide-modified targeted delivery system will overcome the shortcomings of traditional targeted nanomedicine by regulating the components and biological activities of the nanomedicine to form a protein crown in the plasma, and build a blood-brain barrier that can be crossed, while targeting vascular endothelial cells and tumors.
  • the cell's SP-drug complex and SP-modified nano-drug delivery system enable targeted diagnosis and treatment of brain tumors and drug delivery for other brain diseases.
  • An object of the present invention is to provide an amyloid beta short peptide (SP) -mediated brain-targeted delivery system in response to the defects in the prior art, and particularly to a specific apolipoprotein that can be adsorbed in plasma, and Polypeptide modified complexes and targeted delivery systems capable of mediating drugs across the blood-brain barrier and their use in the preparation of preparations for the diagnosis and treatment of peripheral tumors, brain tumors and other brain diseases.
  • the peptide-modified targeted delivery system will overcome the shortcomings of traditional targeted nanomedicine by regulating the components and biological activities of the nanomedicine to form a protein crown in the plasma, and build a blood-brain barrier that can be crossed, while targeting vascular endothelial cells and tumors.
  • the SP-drug complex and SP-modified nano drug delivery system of the cell can realize the targeted diagnosis and treatment of brain tumors (including brain metastases and primary brain tumors) and drug delivery of other brain diseases.
  • SP amyloid beta peptide
  • PVA polylactic acid
  • PLGA refers to: lactic glycolic acid copolymer.
  • PCL polycaprolactone
  • sequence (from N to C terminus) of the SP polypeptide of the present invention is NH 2 -GSNKGAIIGLM-CONH 2 .
  • a first aspect of the present invention provides the use of an amyloid beta short peptide in the preparation of a preparation for mediating drug molecules, fluorescent probes, or delivery systems to target brain tumors and / or other brain lesions, wherein said The amyloid beta short peptide is an amyloid beta short peptide that specifically adsorbs apolipoprotein in plasma; preferably, the brain tumor is a brain metastasis and / or a primary brain tumor.
  • the amyloid ⁇ short peptide is linked to the imaging substance X by a covalent bond to prepare SP-X, which is used for tracing of brain tumors and other disease lesions in the brain;
  • X is a fluorescent molecule Fluorescein or a near-infrared dye molecule Cy5, Cy5.5, Cy7, IR820, ICG, DiR, DiD, DiI;
  • the brain tumor is a brain metastasis and / or a primary brain tumor.
  • SP-Y is prepared by linking the amyloid ⁇ short peptide with an antitumor drug Y by a covalent bond, and is used for targeting brain tumors and other disease lesions in the brain Intervention
  • Y is gefitinib, ectinib, anlotinib, crizotinib, erlotinib, oxitinib, aretinib, paclitaxel, Docetaxel, cabazitaxel, doxorubicin, epirubicin, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin or vincristine small molecule antitumor drugs; and / or
  • Y is a p53 activating peptide or a polypeptide toxin polypeptide antitumor drug
  • the brain tumor is a brain metastasis and / or a primary brain tumor.
  • amyloid ⁇ short peptide is prepared by coupling covalent bonds with polyethylene glycol-Z, and is used to prepare a nano-delivery system.
  • Z is a phospholipid, a polylactic acid, a lactic glycolic acid copolymer, or a polycaprolactone; and / or
  • the nano-delivery system is selected from one or more of the following: a liposome delivery system, a micelle delivery system, a nano-disc delivery system, and a polymer nano-particle delivery system;
  • the SP-polyethylene glycol-phospholipid is used for preparing a liposome delivery system, a micelle delivery system or a nano-disc delivery system; and / or the SP-polyethylene glycol-polylactic acid , SP-polyethylene glycol-lactic acid glycolic acid copolymer, SP-polyethylene glycol-polycaprolactone, used to prepare micellar delivery system and polymer nanoparticle delivery system;
  • the liposome delivery system, micelle delivery system, nano-disc delivery system or polymer nano-particle delivery system is used for encapsulating diagnostic molecules for brain tumors (including brain metastases and primary tumors). Brain tumors) or other diseases of the brain; preferably, the diagnostic system contained in the delivery system is 5-carboxyfluorescein 5-FAM or near-infrared dyes Cy5, Cy5.5, Cy7, IR820, ICG , DiR, DiD, DiI; and / or
  • the nanoparticle delivery system, the liposome delivery system, the micelle delivery system, the polymer nanoparticle delivery system or the nano-disc delivery system are used for encapsulating antitumor drugs for brain tumors (including brain metastases and protozoa). (Primary brain tumor) or targeted intervention of other brain diseases; preferably, the drug contained in the drug delivery system is gefitinib, ectinib, anlotinib, crizotinib, Erlotinib, Oxitinib, Aretinib, Paclitaxel, Docetaxel, Cabazitaxel, Doxorubicin, Epirubicin, Camptothecin, Hydroxycamptothecin, 9-Nitrocamptothecin Base, vincristine, p53 activating peptide or peptide toxin;
  • the brain tumor is a brain metastasis and / or a primary brain tumor.
  • an amyloid beta short peptide for preparing a preparation that mediates a drug molecule, a fluorescent probe, or a delivery system to target brain tumors or other diseases of the brain.
  • the amyloid beta The short peptide is an amyloid beta short peptide that specifically adsorbs apolipoprotein in plasma; preferably, the brain tumor is a brain metastasis and / or a primary brain tumor.
  • a third aspect of the present invention provides a tracer molecule for brain tumors and other disease lesions in the brain.
  • the tracer molecule is the amyloid beta short peptide described in the second aspect with a covalent bond and an imaging substance. X connection to make SP-X;
  • X is a fluorescent molecule Fluorescein or a near-infrared dye molecule Cy5, Cy5.5, Cy7, IR820, ICG, DiR, DiD, DiI;
  • the brain tumor is a brain metastasis and / or a primary brain tumor.
  • a fourth aspect of the present invention provides a medicament for targeted intervention of brain tumors and other disease lesions in the brain, the medicament is the amyloid beta short peptide described in the second aspect with a covalent bond and anti-tumor Drug Y is connected to make SP-Y;
  • Y is gefitinib, ectinib, anlotinib, crizotinib, erlotinib, oxitinib, aretinib, paclitaxel, Docetaxel, cabazitaxel, doxorubicin, epirubicin, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin or vincristine small molecule antitumor drugs; and / or
  • Y is a p53 activating peptide or a polypeptide toxin polypeptide antitumor drug
  • the brain tumor is a brain metastasis and / or a primary brain tumor.
  • a fifth aspect of the present invention provides a nano-delivery system comprising the SP-poly prepared by linking the amyloid beta short peptide of the second aspect with polyethylene glycol-Z by covalent bonding.
  • Z is a phospholipid, a polylactic acid, a lactic glycolic acid copolymer, or a polycaprolactone;
  • the nano-delivery system is selected from one or more of the following: a liposome delivery system, a micelle delivery system, a nano-disc delivery system, and a polymer nano-particle delivery system;
  • the nano delivery system is selected from one or more of the following: a liposome delivery system, a micelle Delivery system and nano-disc delivery system; when the SP-polyethylene glycol-Z is SP-polyethylene glycol-polylactic acid, SP-polyethylene glycol-lactic acid glycolic acid copolymer, SP-polyethylene glycol In the case of alcohol-polycaprolactone, the nano delivery system is a micellar delivery system and / or a polymer nano particle delivery system;
  • the liposome delivery system, micellar delivery system, nano-disc delivery system or polymer nano-particle delivery system is used for encapsulating diagnostic molecules to trace brain tumors or other brain lesions.
  • the diagnostic system contained in the delivery system is 5-carboxyfluorescein 5-FAM or a near-infrared dye Cy5, Cy5.5, Cy7, IR820, ICG, DiR, DiD, DiI; and / or
  • the nanoparticle delivery system, the liposome delivery system, the micelle delivery system, the polymer nanoparticle delivery system or the nano-disc delivery system are used for encapsulating antitumor drugs for brain tumors (including Brain metastases and primary brain tumors) or targeted interventions for other diseases in the brain;
  • the drug contained in the drug delivery system is gefitinib, ectinib, anlotinib, Crizotinib, erlotinib, oxitinib, aretinib, paclitaxel, docetaxel, cabazitaxel, doxorubicin, epirubicin, camptothecin, hydroxycamptothecin, 9 -Nitrocamptothecin, vincristine, p53 activating peptide or polypeptide toxin;
  • the brain tumor is a brain metastasis and / or a primary brain tumor.
  • a sixth aspect of the present invention provides a method for preparing a nano-delivery system according to the fifth aspect, the method comprising the following steps:
  • a seventh aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: 1) the amyloid beta short peptide according to the second aspect, the tracer molecule according to the third aspect, and the The drug and / or nano delivery system according to the fifth aspect; and 2) a pharmaceutically acceptable carrier.
  • An eighth aspect of the present invention provides a diagnostic preparation, the diagnostic preparation comprising: the amyloid beta short peptide according to the second aspect, the tracer molecule according to the third aspect, the drug according to the fourth aspect, and / Or the nano delivery system according to the fifth aspect.
  • a ninth aspect of the present invention provides a method for treating a brain disease, the method comprising: administering to a subject in need the amyloid beta short peptide according to the second aspect, and the third aspect A tracer molecule, a drug according to the fourth aspect, and / or a nano delivery system according to the fifth aspect;
  • the brain disease is a brain tumor or other brain disease
  • the brain tumor is a brain metastasis and / or a primary brain tumor.
  • a tenth aspect of the present invention provides a method for diagnosing brain tumors and other diseases in the brain, the method comprising: administering to a subject in need the amyloid beta short peptide according to the second aspect, The tracer molecule according to the third aspect, the drug according to the fourth aspect, and / or the nano-delivery system according to the fifth aspect; preferably, the brain tumor is a brain metastasis and / or a primary brain tumor.
  • An eleventh aspect of the present invention provides a targeted intervention method for brain tumors or other brain lesions, the method comprising: administering to a subject in need the amyloid beta short peptide according to the second aspect 3.
  • a twelfth aspect of the present invention provides a drug-mediated method, the method comprising: administering to a subject in need the amyloid beta short peptide according to the second aspect, and the short peptide according to the third aspect
  • the tracking molecule, the drug according to the fourth aspect, and / or the nano delivery system according to the fifth aspect preferably, the brain tumor is a brain metastasis and / or a primary brain tumor.
  • amyloid ⁇ in the bionic brain uses apolipoproteins (such as Apoliportein E, Apoliprotein A1, and Apoliprotein J) as molecular chaperones, and the receptor-mediated transport to the peripheral clearance mechanism, and amyloid ⁇ as a template.
  • a short peptide (SP) that specifically binds to the apolipoprotein lipid-binding domain was designed. By modifying the short peptide on the surface of liposomes, it specifically adsorbed apolipoprotein in plasma and maintained its biological properties during blood circulation in vivo.
  • the peptide-modified liposomes can overcome the shortcomings of traditional targeted nanomedicines by regulating the components and biological activities of nanomedicines forming protein crowns in plasma
  • LRP-1 Low-density lipoprotein-related protein 1
  • SRB1 Scavenger receptor class 1 member
  • LRP-2 Low-density lipoprotein-related protein 2
  • the peptide-modified liposomes can overcome the shortcomings of traditional targeted nanomedicines by regulating the components and biological activities of nanomedicines forming protein crowns in plasma
  • SP-drug complex and SP modified nano-drug delivery system that can cross the blood-brain barrier and simultaneously target vascular endothelial cells and tumor cells to achieve targeted diagnosis and treatment of brain tumors and His brain disease of drug delivery.
  • SP is modified by covalent bonds in polyethylene glycol-distearyl phosphatidylethanolamine (PEG-DSPE), polyethylene glycol-polylactic acid (PEG-PLA), and polyethylene glycol-lactic glycolic acid.
  • PEG-DSPE polyethylene glycol-distearyl phosphatidylethanolamine
  • PEG-PLA polyethylene glycol-polylactic acid
  • PEG-lactic glycolic acid polyethylene glycol-lactic glycolic acid.
  • Copolymers PEG-PLGA
  • PEG-PCL polyethylene glycol-polycaprolactone
  • other materials were used to construct delivery systems such as SP short peptide-modified liposomes, micelles, nanodiscs, and nanoparticles.
  • the SP-modified nano delivery system can contain gefitinib, ectinib, anlotinib, crizotinib, erlotinib, oxitinib, aretinib, paclitaxel, and more Sitaxel, cabazitaxel, doxorubicin, epirubicin, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, vincristine, p53 activating peptide, melittin, scorpion peptide, etc.
  • Antitumor drugs; and, can contain fluorescent substances, such as FAM, near-infrared dyes Cy5, Cy5.5, Cy7, IR820, ICG, DiR, DiD, DiI, and so on.
  • SP-modified drugs or probes include the formation of pH-sensitive hydrazone bonds through the reaction of maleimide hexazide derivatives, involving drugs such as doxorubicin, epirubicin, p53 activating peptide, polypeptide toxin, or the like 3- (2-pyridine dimercapto) propionic acid derivative reacts to form a disulfide bond, involving gefitinib, ectinib, anlotinib, crizotinib, erlotinib, oxitinib, Aretinib, paclitaxel, docetaxel, cabazitaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, vincristine, p53 activating peptide, peptide toxin and other drugs, or through stable chemical bonds
  • fluorescent probes involving Fluorescein, Cy5, Cy5.5, Cy7, ICG, IR820.
  • amyloid ⁇ short peptides for preparing brain tumors (including brain metastases and primary brain tumors) or other brain lesions in the preparation of mediating drug molecules, fluorescent probes or delivery systems.
  • the amyloid beta short peptide SP is an amyloid beta short peptide that specifically adsorbs apolipoprotein in plasma.
  • the amyloid beta short peptide is connected to the imaging substance X by a covalent bond to prepare SP-X, which is used for brain tumors (including brain metastases and primary brain tumors) and other diseases in the brain.
  • the amyloid beta short peptide is linked with an antitumor drug Y by a covalent bond to prepare SP-Y, which is used for brain tumors (including brain metastases and primary brain tumors) and other diseases in the brain.
  • Y is gefitinib, ectinib, anlotinib, crizotinib, erlotinib, oxitinib, aretinib , Paclitaxel, docetaxel, cabazitaxel, doxorubicin, epirubicin, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin or vincristine small molecule antitumor drugs; said In SP-Y, Y is a p53 activating peptide or a peptide toxin polypeptide antitumor drug;
  • the SP-polyethylene glycol-Z prepared by linking amyloid beta short peptide (SP) with polyethylene glycol-Z by a covalent bond is used to prepare a nano delivery system;
  • the SP-polyethylene In the diol-Z, Z is a phospholipid, a polylactic acid (PLA), a lactic glycolic acid copolymer (PLGA), or a polycaprolactone (PCL);
  • the SP-polyethylene glycol-phospholipid described in the present invention is used to prepare a liposome delivery system, a micelle delivery system or a nano-disc delivery system;
  • SP-polyethylene glycol-polylactic acid, SP-polyethylene glycol-lactic acid glycolic acid copolymer, and SP-polyethylene glycol-polycaprolactone described in the present invention are used for preparing a micelle delivery system and polymerization.
  • the liposome delivery system, micelle delivery system, nano-disc delivery system or polymer nano-particle delivery system described in the present invention is used for encapsulating diagnostic molecules for brain tumors (including brain metastases and primary brain Tumor) or other brain lesions;
  • the diagnostic system contained in the delivery system is 5-carboxyfluorescein 5-FAM or near-infrared dyes Cy5, Cy5.5, Cy7, IR820, ICG, DiR, DiD , DiI;
  • the nanoparticle delivery system, liposome delivery system, micelle delivery system, polymer nanoparticle delivery system or nano-disc delivery system described in the present invention is used for encapsulating antitumor drugs for brain tumors (including brain metastases). Tumors and primary brain tumors) or targeted interventions for other brain lesions; the drugs included are gefitinib, ectinib, anlotinib, crizotinib, erlotinib, Austria Citrinib, aretinib, paclitaxel, docetaxel, cabazitaxel, doxorubicin, epirubicin, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, vincristine, p53 activates a peptide or polypeptide toxin.
  • the cysteine thiol-containing SP was reacted with maleimide-PEG-DSPE in a phosphate buffer at pH 7.4, and the product was lyophilized after purification to prepare SP-PEG-DSPE for future use.
  • the binding activity to the LRP-1 receptor was detected by western blot and radiolabeling methods.
  • the SP-modified delivery system and the delivery of unmodified SP that were not incubated with serum The system serves as a negative control.
  • Vascular endothelial cells (such as bEND.3 cells) were used to compare the uptake of SP-modified and unmodified delivery systems to endothelial cells before and after serum incubation.
  • mice such as Kunming, C57BL / 6, etc.
  • a fluorescein-loaded delivery system in the tail vein to compare the accumulation of SP modified and unmodified delivery systems in the brain of mice at different time points.
  • Lipid-A-containing polypeptide modified and unmodified delivery systems were prepared. They were injected intraperitoneally into Balb / c mice every four days for four weeks, and blood was collected from the orbit on the seventh day after each injection. ELISA was used to detect the IgG and IgM content against PEG and SP in mice at different time points to evaluate the immunogenicity.
  • the test of the present invention shows that the SP-mediated drug or drug delivery system targets cells and tissues with high expression of LRP-1, and has the ability to cross the biological membrane barrier, especially the ability to cross the blood-brain barrier (BBB). It can be used for targeted diagnosis and treatment of brain tumors (including brain metastases and primary brain tumors) and other diseases.
  • BBB blood-brain barrier
  • the experiments of the present invention show that SP is modified on the surface of the drug delivery system, and combines with apolipoprotein in plasma to increase the uptake of vascular endothelial cells after forming a protein crown; SP can simultaneously mediate drugs across the blood-brain barrier and / or target tumor neoplasia Blood vessels and tumor cells, and their modified drugs and drug delivery systems can achieve better therapeutic effects in the treatment of brain tumors (including brain metastases and primary brain tumors) and other diseases in the brain.
  • SP was modified by chemical coupling to obtain SP-sLip on the surface of liposomes, mixed with mouse plasma and incubated at 37 ° C for 1 h, and then centrifuged at high speed to obtain a liposome precipitate containing a protein crown.
  • the analysis of plasma protein components adsorbed by liposomes was obtained by rapid silver staining (as shown in Figure 1A).
  • the results showed that compared with methoxypolyethylene glycol modified long-circulating liposomes (sLip), SP-sLip adsorbed plasma proteins at 45KDa (band 1), 38KDa (band 2), and 25KDa (band). Bands at 3) increased significantly.
  • the PAGE gels corresponding to the same three positions of the two were analyzed by LC-MS / MS and western blot (shown in FIG. 1B), and the proteins in the three positions were identified as mouse-derived ApoJ, ApoE, and ApoA1.
  • Western blot analysis also confirmed that SP-sLip significantly increased the three apolipoproteins compared to sLip adsorption (as shown in Figures 1C-E).
  • SP-sLip The SP-modified liposome itself can specifically adsorb the recombinant protein LRP-1 in solution (as shown in FIG. 3A).
  • FIG. 3A We confirmed by western blot method that after SP-sLip was pre-incubated with ApoE, it still retained the ability to bind to LRP-1 (as shown in Figures 3B-C).
  • the 3DE shown in Figure 3DE demonstrates that both free SP and modified liposomes (SP-sLip) can compete with SP-antibodies immobilized on 96-well plates, and SP-sLip loses competitive antibody binding after plasma incubation.
  • Apolipoproteins such as ApoE that specifically bind to the surface of SP-sLip have the activity of binding to the BBB receptor LRP-1, becoming an effective way to mediate SP-sLip across the BBB into the brain.
  • SP-sLip SP-modified liposomes
  • SP modification on the surface of liposomes did not affect the pharmacokinetic parameters of liposomes in vivo (as shown in Figure 5A), nor did it increase the immunogenicity of ordinary long-circulating liposomes (in blood IgG and IgM are evaluation indicators, as shown in Figures 5B-D).
  • Figure 6 Evaluation of SP entry liposomes in the brain in mice
  • the median survival time of the mice in the saline group, the DOX group, the sLip / DOX group, and the SP-sLip / DOX group were 27 days, 31 days, 33 days, and 50 days, respectively.
  • SP-modified liposomes could significantly extend the brain Median survival of orthotopic tumor model mice.
  • SP-sLip / DOX SP modified liposome-encapsulated doxorubicin group
  • SP-sLip / DOX SP modified liposome-encapsulated doxorubicin group
  • SP did not show cytotoxicity on the in vitro cultured nerve cell line PC12 (as shown in Figure 10A), and SP modified liposomes encapsulated the organs of the adriamycin group (SP-sLip / DOX) mice. There were no obvious abnormalities in the tissue sections (as shown in FIG. 10B), indicating that the SP modification did not cause toxic and side effects on the surface of the liposome.
  • Preparation of liposome SP-sLip / DiI Weigh 50mg Mal-PEG-DSPE and dissolve it in 5mL CHCl 3 at 37 ° C to form a film. After vacuum drying for half an hour, dissolve in 4mL double distilled water, hydrate at 37 ° C, and sonicate to remove Large particles. Weigh 26mg of SP-Cys protein in 2mL of double-distilled water, mix it with the solution of the membrane material, and rinse the container with 1mL of double-distilled water.
  • HSPC 7.85mg cholesterol: 3.35mg; mPEG-2000-DSPE: 1.67mg; SP-PEG-DSPE: 1.82mg; DiI: 0.4mg, dissolved in 10mL CHCl 3 , 40 degrees
  • the film was suspended in a water bath to form a film.
  • the organic solvent was removed by vacuum drying.
  • the film was dissolved in 1 mL of double-distilled water to obtain a 60-degree water bath and shaken to hydrate.
  • the liposome extruder was extruded at 400nm, 200nm, and 100nm to obtain SP-sLip. / DiI;
  • Preparing liposomes sLip / DOX Weigh membrane taken HSPC liposomes prepared: 7.85mg Cholesterol: 3.35mg; mPEG-2000-DSPE : 2.78mg, was dissolved in 10mL CHCl 3, 40 ° water bath deposition suspension was evaporated The organic solvent was removed by vacuum drying. The membrane was dissolved in 1 mL of ammonium sulfate solution (0.32M) to obtain a 60-degree water bath and shaken to hydrate. The liposome extruder was extruded at a pore diameter of 400 nm, 200 nm, and 100 nm to obtain an ammonium sulfate solution.
  • the hydrated sLip was replaced with a normal saline solvent by a G50 elution column, and an adriamycin aqueous solution was added and mixed (drug-to-lipid ratio 1:10), and the unencapsulated doxorubicin was removed by a G50 chromatography column to obtain sLip / DOX;
  • Example 2 Binding activity of liposomes to LRP-1 receptors after formation of a protein crown in serum and its effect on uptake by vascular endothelial cells
  • C57BL / 6 mouse serum (containing protease inhibitor, EDTA as anticoagulant) was mixed with liposomes 1: 1, incubated at 37 ° C for 1 h, centrifuged at 15000 rpm for 30 min, washed twice with cold PBS and dissolved in In 30 ⁇ L PBS, use serum as a positive control and PBS as a negative control.
  • the in vivo experiment is performed as follows: Fluorescent DiI-labeled peptide-modified and unmodified liposomes are injected into C57BL / 6 mice through the tail vein. Blood is taken and plasma is centrifuged at low speed after 1 hour. The protein crown components adsorbed in vivo were identified by SDS-PAGE and western blot methods, as shown in Figure 2.
  • ELISA method was used to detect the binding activity of SP-sLip and SP antibody before and after serum incubation to determine the change in the active binding domain of SP and receptor after plasma protein interaction, and to detect its apolipoprotein activity.
  • the specific operation is as follows: ELISA plate Add 0.1 ⁇ g of SP-PEG-DSPE to each well, and overnight at room temperature. After washing 3 times with PBS, block with 3% BSA for 1 h. Aspirate the BSA solution, add SP antibody and incubate at 37 ° C for 1 h.
  • liposomes incubated with PBS and gradient dilution incubated at 37 ° C for 1 hour, washed three times with PBS, added the corresponding horseradish peroxidase-labeled secondary antibody, and reacted with the TMB chromogenic solution for 3-15min after 1h.
  • H 2 SO 4 terminated the reaction, and its absorbance was measured at a wavelength of 450 nm.
  • the experimental results are shown in FIG. 3.
  • Resuscitation of vascular endothelial cells bEND3 The frozen bEND3 cells were quickly thawed and transferred to a centrifuge tube pre-added with culture medium. After centrifugation at 1000 r / min for 3 minutes, the supernatant was discarded, and DMEM culture solution containing 10% FBS was added and gently blown. Add evenly to a petri dish and mix well. Observe cell morphology and growth under a microscope and culture in an incubator at 37 ° C, 5% CO 2 and 95% relative humidity;
  • vascular endothelial cells bEND3 culture of vascular endothelial cells bEND3 cells, that is, the number of cells, morphology, and adherence. Aspirate the old culture medium, add fresh DMEM medium containing 10% FBS, mix well, continue to culture in an incubator at 37 ° C, 5% CO 2 , and saturated humidity, and observe the cell growth every day, every 2-3 Passage by day, about 10 days or so, the cells are in the logarithmic growth phase and can be used for in vitro cell experiments;
  • vascular endothelial cells bEND3 Passage of vascular endothelial cells bEND3: aspirate and discard the culture solution, rinse it twice with PBS, add a little 0.25% trypsin, place it in the incubator for 1 min, and then add 2-3 mL of culture solution to terminate the digestion. Divide the cell suspension into equal portions. Centrifuge tubes, discard the supernatant after centrifugation, add new culture medium, transfer to a petri dish, and place in an incubator for cultivation;
  • Cryopreservation of vascular endothelial cells bEND3 After the end of the cell experiment, freeze the cells for next use. Prepare a cryopreservation solution (containing 10% DMSO and 90% fetal bovine serum) in advance and pre-chill at 4 ° C. After digesting the cells with trypsin, add the pre-chilled cryopreservation solution, and gently mix by pipetting. Add 1mL of cell liquid to each cryopreservation tube, seal the name of the cryopreserved cells and the date of the cryopreservation after sealing, set at -80 ° C overnight, save in a liquid nitrogen tank and register for the record;
  • a cryopreservation solution containing 10% DMSO and 90% fetal bovine serum
  • BEND3 of appropriate density was inoculated into a 6-well plate, cultured in a cell incubator overnight, and fluorescein DiI-labeled liposomes (serum-incubated and non-incubated with serum) were separately used in serum-free DMEM medium. Dilute 50 times, add 1ml to each well, and incubate with the cells for 4h. Aspirate the culture solution, wash it twice with PBS, trypsinize the cells, digest the cells with serum-containing medium, centrifuge, and wash with PBS. Finally, the cells are dispersed.
  • Example 3 SP-modified liposomes can span the BBB
  • SP modified liposomes SP-sLip / DiI labeled with fluorescein DiI can cross the blood-brain barrier:
  • nude mouse model of glioma take U87 cells in logarithmic growth phase, digest the cells and count them, and suspend them with an appropriate amount of PBS buffer. Each nude mouse is inoculated with 6 ⁇ 10 5 cells (dispersed in 5 ⁇ L PBS buffer). (Middle), the nude mice were anesthetized with 7% chloral hydrate before the experiment, and fixed with a brain stereotaxic device, and the suspended cells were seeded in the striatum (ie, the anterior condyle 0.6 mm forward, 1.8 mm to the right, and 3 mm deep) , Regularly observe the status of nude mice after surgery;
  • Doxorubicin-containing SP-sLip inhibits neovascularization and tumor cell apoptosis in tumors:
  • the orthotopic glioma model mice were anesthetized with chloral hydrate, the brain tissue was separated, fixed with paraformaldehyde, dehydrated, and paraffin-embedded sections.
  • CD31 antibody was used to detect the inhibitory effect on neovascularization by immunofluorescence staining, and the terminal deoxynucleotidyl transferase (TDT) -mediated dUTP gap terminal labeling method (Terminal deoxynucleotidyl Transferase-mediated UTP, Nick end labeling, TUNEL) was used to detect tumor The degree of cell apoptosis was observed and photographed by a confocal fluorescence microscope. The experimental results are shown in Figures 8 and 9.

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Abstract

一种药学领域可特异性吸附血浆中载脂蛋白，且能介导药物跨血脑屏障的多肤修饰的复合物和靶向递送系统及其在制备诊断与治疗脑肿瘤及其它脑部疾病的制剂中的应用。所述的淀粉样蛋白β(amyloid，涉及一种)的多肽片段(SP)经共价键修饰荧光探针、药物分子和脂质体递药系统，经试验显示，所修饰的递送系统在与血浆蛋白作用形成蛋白冠后，增加其被血管内皮细胞的摄取；所修饰的脂质体递送系统更有效的将药物递送至病灶部位，显著提高药物治疗效果。所述的SP吸附血浆蛋白后，可介导药物跨血脑屏障和/或靶向肿瘤新生血管和肿瘤细胞，其修饰的药物和递送系统在治疗脑肿瘤及脑内其它疾病时获得更好的治疗效果。

Description

一种淀粉样蛋白β短肽介导的脑靶向递送系统及其制备方法和用途
相关申请的交叉引用
本申请要求2018年06月19日提交的第CN201810626652.4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，涉及一种淀粉样蛋白β短肽(SP)介导的脑靶向递送系统，尤其涉及一种可特异性吸附血浆中载脂蛋白，且能介导药物跨血脑屏障的多肽修饰的复合物和靶向递送系统及其在制备诊断与治疗脑肿瘤(含脑转移瘤和原发性脑肿瘤)及其它脑部疾病的制剂中的应用。
背景技术
现有技术公开了中枢神经系统疾病已成为严重危害人类健康的疾患，但相关新药的研发进展仍十分缓慢，其主要原因之一是所述药物中的绝大部分药物分子难以透过血－脑屏障，而设计可跨血－脑屏障的药物或递药系统乃是目前药物研发领域的重点和难点之一。研究公开了脑靶向递药系统主要利用受体介导方式跨越血脑屏障入脑，但目前的有关药物设计多为侧重于脑靶向分子与血脑屏障上相关受体的结合效率，常忽略了递药系统体内传输过程中体液成分的影响，如：结合血浆蛋白形成蛋白冠可极大影响递药系统的靶向性和体内循环时间等，因此，导致体外设计的脑靶向递药系统在体内应用中靶向效率欠佳。如何从全新的角度设计脑靶向递药系统，精准调控其在体内传输过程中与相关蛋白或细胞的相互作用，包括：血浆蛋白吸附和解离、其他细胞吞噬作用、蛋白降解、免疫反应等，发挥更有效的靶向功能，是当前业内研究的热点。
基于现有技术的现状，本申请的发明人拟提供一种淀粉样蛋白β短肽(下文简称为SP)介导的脑靶向递送系统，尤其涉及一种可特异性吸附血浆中载脂蛋白，且能介导药物跨血脑屏障的多肽修饰的复合物和靶向递送系统及其在制备诊断与治疗脑肿瘤(含脑转移瘤和原发性脑肿瘤)及其它脑部疾病的制剂中的应用。该多肽修饰的靶向递送系统将通过调控纳米药物在血浆中形成蛋白冠的组分和生物活性，克服传统靶向纳米药物的缺陷，构建可跨血脑屏障，同时靶向血管内皮细胞、肿瘤细胞的SP-药物复合物和SP修饰的纳米递药系统，实现脑肿瘤的靶向诊断和治疗及其他脑部疾病的药物递送。
发明内容
本发明的目的在于，针对现有技术存在的缺陷，提供一种淀粉样蛋白β短肽(SP) 介导的脑靶向递送系统，尤其涉及一种可特异性吸附血浆中载脂蛋白，且能介导药物跨血脑屏障的多肽修饰的复合物和靶向递送系统及其在制备诊断与治疗外周肿瘤、脑肿瘤及其它脑部疾病的制剂中的应用。该多肽修饰的靶向递送系统将通过调控纳米药物在血浆中形成蛋白冠的组分和生物活性，克服传统靶向纳米药物的缺陷，构建可跨血脑屏障，同时靶向血管内皮细胞、肿瘤细胞的SP-药物复合物和SP修饰的纳米递药系统，实现脑肿瘤(含脑转移瘤和原发性脑肿瘤)的靶向诊断和治疗及其他脑部疾病的药物递送。
在阐述本发明内容之前，定义本文中所使用的术语如下：
术语“SP”是指：淀粉样蛋白β短肽。
术语“PLA”是指：聚乳酸。
术语“PLGA”是指：乳酸羟基乙酸共聚物。
术语“PCL”是指：聚己内酯。
本发明SP多肽的序列(从N到C端)为NH 2-GSNKGAIIGLM-CONH 2。
本发明的第一方面提供了淀粉样蛋白β短肽在制备用于介导药物分子、荧光探针或递送系统靶向脑肿瘤和/或脑部其它疾病病灶的制剂中的应用，其中所述的淀粉样蛋白β短肽为特异性吸附血浆中载脂蛋白的淀粉样蛋白β短肽；优选地，所述脑肿瘤为脑转移瘤和/或原发性脑肿瘤。
根据本发明第一方面的用途，其中，所述的淀粉样蛋白β短肽以共价键与影像物质X连接制得SP-X，用于脑肿瘤及脑内其它疾病病灶的示踪；
优选地，所述的SP-X中，X是荧光分子Fluorescein或近红外染料分子Cy5、Cy5.5、Cy7、IR820、ICG、DiR、DiD、DiI；
更优选地，所述脑肿瘤为脑转移瘤和/或原发性脑肿瘤。
根据本发明第一方面的用途，其中，按所述的淀粉样蛋白β短肽以共价键与抗肿瘤药物Y连接制得SP-Y，用于脑肿瘤及脑内其它疾病病灶的靶向干预；
优选地，所述的SP-Y中，Y是吉非替尼、埃克替尼、安罗替尼、克唑替尼、厄洛替尼、奥希替尼、阿雷替尼、紫杉醇、多西他赛、卡巴他赛、阿霉素、表阿霉素、喜树碱、羟基喜树碱、9-硝基喜树碱或长春新碱小分子抗肿瘤药物；和/或
所述的SP-Y中，Y是p53激活肽或多肽毒素多肽抗肿瘤药物；
更优选地，所述脑肿瘤为脑转移瘤和/或原发性脑肿瘤。
根据本发明第一方面的用途，其中，所述的淀粉样蛋白β短肽用共价键与聚乙二醇 -Z连接制得的SP-聚乙二醇-Z，用于制备纳米递送系统；
优选地，所述的SP-聚乙二醇-Z中，Z是磷脂、聚乳酸、乳酸羟基乙酸共聚物或聚己内酯；和/或
优选地，所述的纳米递送系统选自以下一种或多种：脂质体递送系统、胶束递送系统、纳米圆盘递送系统和和聚合物纳米粒递送系统；
更优选地，所述的SP-聚乙二醇-磷脂用于制备脂质体递送系统、胶束递送系统或纳米圆盘递送系统；和/或所述的SP-聚乙二醇-聚乳酸、SP-聚乙二醇-乳酸羟基乙酸共聚物、SP-聚乙二醇-聚己内酯，用于制备胶束递送系统和聚合物纳米粒递送系统；
进一步优选地，所述的脂质体递送系统、胶束递送系统、纳米圆盘递送系统或聚合物纳米粒递送系统，用于包载诊断分子，进行脑肿瘤(含脑转移瘤和原发性脑肿瘤)或脑部其它疾病病灶的示踪；优选地，所述的递送系统所包载诊断分子是5-羧基荧光素5-FAM或近红外染料Cy5、Cy5.5、Cy7、IR820、ICG、DiR、DiD、DiI；和/或
所述的纳米粒递送系统、脂质体递送系统、胶束递送系统、聚合物纳米粒递送系统或纳米圆盘递送系统，用于包载抗肿瘤药物，进行脑肿瘤(含脑转移瘤和原发性脑肿瘤)或脑部其它疾病病灶的靶向干预；优选地，所述的递药系统所包载药物是吉非替尼、埃克替尼、安罗替尼、克唑替尼、厄洛替尼、奥希替尼、阿雷替尼、紫杉醇、多西他赛、卡巴他赛、阿霉素、表阿霉素、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、p53激活肽或多肽毒素；
更进一步优选地，所述脑肿瘤为脑转移瘤和/或原发性脑肿瘤。
本发明的第二方面提供了一种用于制备介导药物分子、荧光探针或递送系统靶向脑肿瘤或脑部其它疾病病灶制剂的淀粉样蛋白β短肽，所述的淀粉样蛋白β短肽为特异性吸附血浆中载脂蛋白的淀粉样蛋白β短肽；优选地，所述脑肿瘤为脑转移瘤和/或原发性脑肿瘤。
本发明的第三方面提供了一种用于脑肿瘤及脑内其它疾病病灶的示踪分子，所述示踪分子为第二方面所述的淀粉样蛋白β短肽以共价键与影像物质X连接制得SP-X；
优选地，所述的SP-X中，X是荧光分子Fluorescein或近红外染料分子Cy5、Cy5.5、Cy7、IR820、ICG、DiR、DiD、DiI；
更优选地，所述脑肿瘤为脑转移瘤和/或原发性脑肿瘤。
本发明的第四方面提供了一种用于脑肿瘤及脑内其它疾病病灶的靶向干预的药物， 所述药物为第二方面所述的淀粉样蛋白β短肽以共价键与抗肿瘤药物Y连接制得SP-Y；
优选地，所述的SP-Y中，Y是吉非替尼、埃克替尼、安罗替尼、克唑替尼、厄洛替尼、奥希替尼、阿雷替尼、紫杉醇、多西他赛、卡巴他赛、阿霉素、表阿霉素、喜树碱、羟基喜树碱、9-硝基喜树碱或长春新碱小分子抗肿瘤药物；和/或
所述的SP-Y中，Y是p53激活肽或多肽毒素多肽抗肿瘤药物；
更优选地，所述脑肿瘤为脑转移瘤和/或原发性脑肿瘤。
本发明的第五方面提供了一种纳米递送系统，所述纳米递送系统包含第二方面所述的淀粉样蛋白β短肽用共价键与聚乙二醇-Z连接制得的SP-聚乙二醇-Z；
优选地，所述的SP-聚乙二醇-Z中，Z是磷脂、聚乳酸、乳酸羟基乙酸共聚物或聚己内酯；
更优选地，所述的纳米递送系统选自以下一种或多种：脂质体递送系统、胶束递送系统、纳米圆盘递送系统和和聚合物纳米粒递送系统；
进一步优选地，当所述的SP-聚乙二醇-Z为SP-聚乙二醇-磷脂时，所述的纳米递送系统选自以下一种或多种：脂质体递送系统、胶束递送系统和纳米圆盘递送系统；当所述的SP-聚乙二醇-Z为SP-聚乙二醇-聚乳酸、SP-聚乙二醇-乳酸羟基乙酸共聚物、SP-聚乙二醇-聚己内酯时，所述的纳米递送系统为胶束递送系统和/或聚合物纳米粒递送系统；
进一步优选地，所述的脂质体递送系统、胶束递送系统、纳米圆盘递送系统或聚合物纳米粒递送系统，用于包载诊断分子，进行脑肿瘤或脑部其它疾病病灶的示踪；优选地，所述的递送系统所包载诊断分子是5-羧基荧光素5-FAM或近红外染料Cy5、Cy5.5、Cy7、IR820、ICG、DiR、DiD、DiI；和/或
进一步优选地，所述的纳米粒递送系统、脂质体递送系统、胶束递送系统、聚合物纳米粒递送系统或纳米圆盘递送系统，用于包载抗肿瘤药物，进行脑部肿瘤(含脑转移瘤和原发性脑肿瘤)或脑部其它疾病病灶的靶向干预；优选地，所述的递药系统所包载药物是吉非替尼、埃克替尼、安罗替尼、克唑替尼、厄洛替尼、奥希替尼、阿雷替尼、紫杉醇、多西他赛、卡巴他赛、阿霉素、表阿霉素、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、p53激活肽或多肽毒素；
更进一步优选地，所述脑肿瘤为脑转移瘤和/或原发性脑肿瘤。
本发明的第六方面提供了第五方面所述的纳米递送系统的制备方法，所述方法包括以下步骤：
称取天然磷脂，胆固醇，甲氧基-PEG-DSPE，与SP-聚乙二醇-Z，模型药物或探针分子，溶解于溶媒中，成膜，水化，过膜，柱层析除去游离探针分子或药物，制得所述纳米递送系统；和/或
称取天然磷脂，胆固醇，甲氧基-PEG-DSPE，与SP-聚乙二醇-Z溶解于溶媒中，成膜，水化，过膜，主动包封探针分子或药物，柱层析除去游离探针分子或药物，制得所述纳米递送系统。
本发明的第七方面提供了一种药物组合物，所述药物包含：1)第二方面所述的淀粉样蛋白β短肽、第三方面所述的示踪分子、第四方面所述的药物和/或第五方面所述的纳米递送系统；以及2)药学上可接受的载体。
本发明的第八方面提供了一种诊断制剂，所述诊断制剂包含：第二方面所述的淀粉样蛋白β短肽、第三方面所述的示踪分子、第四方面所述的药物和/或第五方面所述的纳米递送系统。
本发明的第九方面提供了一种用于治疗脑部疾病的方法，所述方法包括：对有需要的受试者给予第二方面所述的淀粉样蛋白β短肽、第三方面所述的示踪分子、第四方面所述的药物和/或第五方面所述的纳米递送系统；
优选地，所述脑部疾病为脑肿瘤或脑部其它疾病；
更优选地，所述脑肿瘤为脑转移瘤和/或原发性脑肿瘤。
本发明的第十方面提供了一种用于诊断脑肿瘤及脑内其它疾病病灶的方法，所述方法包括：对有需要的受试者给予第二方面所述的淀粉样蛋白β短肽、第三方面所述的示踪分子、第四方面所述的药物和/或第五方面所述的纳米递送系统；优选地，所述脑肿瘤为脑转移瘤和/或原发性脑肿瘤。
本发明的第十一方面提供了一种脑肿瘤或脑部其它疾病病灶的靶向干预方法，所述方法包括：对有需要的受试者给予第二方面所述的淀粉样蛋白β短肽、第三方面所述的示踪分子、第四方面所述的药物和/或第五方面所述的纳米递送系统；优选地，所述脑肿瘤为脑转移瘤和/或原发性脑肿瘤。
本发明的第十二方面提供了一种一种药物介导方法，所述方法包括：对有需要的受试者给予第二方面所述的淀粉样蛋白β短肽、第三方面所述的示踪分子、第四方面所述的药物和/或第五方面所述的纳米递送系统；优选地，所述脑肿瘤为脑转移瘤和/或原发性脑肿瘤。
本发明中，仿生脑内淀粉样蛋白β以载脂蛋白(如Apoliportein E、Apoliprotein A1和Apoliprotein J)为分子伴侣、经受体介导转运至外周的清除机制，以淀粉样蛋白β为 模板，设计了可特异性结合载脂蛋白脂质结合域的短肽(SP)，通过将该短肽修饰在脂质体表面，在体内血液循环过程中特异性吸附血浆中载脂蛋白并保持其生物活性，进而利用载脂蛋白与血脑屏障上多种受体[包括Low-density lipoprotein-related protein 1(LRP-1)，Scavenger receptor class B member 1(SRB1)和Low-density lipoprotein-related protein 2(LRP-2)]结合，介导脂质体转运入脑；该多肽修饰的脂质体通过调控纳米药物在血浆中形成蛋白冠的组分和生物活性，能克服传统靶向纳米药物的缺陷，构建可跨血脑屏障，同时靶向血管内皮细胞、肿瘤细胞的SP-药物复合物和SP修饰的纳米递药系统，实现脑肿瘤的靶向诊断和治疗及其他脑部疾病的药物递送。
本发明中，SP以共价键修饰在聚乙二醇-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)等材料上，构建SP短肽修饰的脂质体、胶束、纳米圆盘、纳米粒等递送系统。
所述的SP修饰的纳米递送系统可包载吉非替尼、埃克替尼、安罗替尼、克唑替尼、厄洛替尼、奥希替尼、阿雷替尼、紫杉醇、多西他赛、卡巴他赛、阿霉素、表阿霉素、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、p53激活肽、蜂毒肽、蝎毒肽等抗肿瘤药物；以及，可包载荧光物质，如FAM、近红外染料Cy5、Cy5.5、Cy7、IR820、ICG、DiR、DiD、DiI等。
本发明中，SP修饰的药物或探针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、p53激活肽、多肽毒素等药物、或通过3-(2-吡啶二巯基)丙酸衍生物反应形成二硫键，涉及吉非替尼、埃克替尼、安罗替尼、克唑替尼、厄洛替尼、奥希替尼、阿雷替尼、紫杉醇、多西他赛、卡巴他赛、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、p53激活肽、多肽毒素等药物、或通过稳定化学键连接荧光探针，涉及Fluorescein、Cy5、Cy5.5、Cy7、ICG、IR820。
进一步的，本发明提供了淀粉样蛋白β短肽在制备介导药物分子、荧光探针或递送系统靶向脑肿瘤(含脑转移瘤和原发性脑肿瘤)或脑部其它疾病病灶制剂中的应用，所述的淀粉样蛋白β短肽(SP)为特异性吸附血浆中载脂蛋白的淀粉样蛋白β短肽。
所述的淀粉样蛋白β短肽(SP)以共价键与影像物质X连接制得SP-X，用于脑肿瘤(含脑转移瘤和原发性脑肿瘤)及脑内其它疾病病灶的示踪；所述的SP-X中，X是荧光分子Fluorescein或近红外染料分子Cy5、Cy5.5、Cy7、IR820、ICG、DiR、DiD、DiI；
所述的淀粉样蛋白β短肽(SP)以共价键与抗肿瘤药物Y连接制得SP-Y，用于脑肿瘤(含脑转移瘤和原发性脑肿瘤)及脑内其它疾病病灶的靶向干预中；所述的SP-Y中，Y是吉非替尼、埃克替尼、安罗替尼、克唑替尼、厄洛替尼、奥希替尼、阿雷替尼、紫杉醇、多西他赛、卡巴他赛、阿霉素、表阿霉素、喜树碱、羟基喜树碱、9-硝基喜树碱或长春新碱小分子抗肿瘤药物；所述的SP-Y中，Y是p53激活肽或多肽毒素多肽抗肿 瘤药物；
所述的淀粉样蛋白β短肽(SP)用共价键与聚乙二醇-Z连接制得的SP-聚乙二醇-Z，用于制备纳米递送系统；所述的SP-聚乙二醇-Z中，Z是磷脂、聚乳酸(PLA)、乳酸羟基乙酸共聚物(PLGA)或聚己内酯(PCL)；
本发明中所述的SP-聚乙二醇-磷脂用于制备脂质体递送系统、胶束递送系统或纳米圆盘递送系统；
本发明中所述的SP-聚乙二醇-聚乳酸、SP-聚乙二醇-乳酸羟基乙酸共聚物、SP-聚乙二醇-聚己内酯，用于制备胶束递送系统和聚合物纳米粒递送系统；
本发明中所述的脂质体递送系统、胶束递送系统、纳米圆盘递送系统或聚合物纳米粒递送系统，用于包载诊断分子，进行脑肿瘤(含脑转移瘤和原发性脑肿瘤)或脑部其它疾病病灶的示踪；所述的递送系统所包载诊断分子是5-羧基荧光素5-FAM或近红外染料Cy5、Cy5.5、Cy7、IR820、ICG、DiR、DiD、DiI；
本发明中所述的纳米粒递送系统、脂质体递送系统、胶束递送系统、聚合物纳米粒递送系统或纳米圆盘递送系统，用于包载抗肿瘤药物，进行脑肿瘤(含脑转移瘤和原发性脑肿瘤)或脑部其它疾病病灶的靶向干预；所包载药物是吉非替尼、埃克替尼、安罗替尼、克唑替尼、厄洛替尼、奥希替尼、阿雷替尼、紫杉醇、多西他赛、卡巴他赛、阿霉素、表阿霉素、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、p53激活肽或多肽毒素。
更具体的，本发明通过下述技术方案实现，
1.制备SP修饰的脂质体递送系统
将含半胱氨酸巯基的SP与马来酰亚胺-PEG-DSPE在PH 7.4的磷酸缓冲液中反应，纯化后冻干产物，制备SP-PEG-DSPE备用，
称取一定量的天然磷脂，胆固醇，甲氧基-PEG-DSPE(mPEG-DSPE)，与SP-PEG-DSPE，模型药物或探针分子，溶解于溶媒中，成膜，水化，脂质体挤压器过膜，柱层析除去游离探针分子或药物，制得SP多肽修饰的脂质体，动态光散射粒度仪表征其粒径和表面电位。或称取一定量的天然磷脂，胆固醇，甲氧基-PEG-DSPE(mPEG-DSPE)，与SP-PEG-DSPE，溶解于溶媒中，成膜，水化，脂质体挤压器过膜，主动包封探针分子或药物，柱层析除去游离探针分子或药物，制得SP修饰的脂质体，动态光散射粒度仪表征其粒径和表面电位。
2.SP修饰的递送系统在与血浆共孵育形成蛋白冠后对其结合活性的影响实验，
将SP修饰的递送系统与新鲜血浆共孵育一段时间后，用western blot和放射性标记方法检测其与LRP-1受体的结合活性，未与血清孵育的SP修饰的递送系统和未修饰SP的递送系统作为阴性对照。
3.SP修饰对递送系统被血管内皮细胞系统摄取的影响实验，
用血管内皮细胞(如bEND.3细胞)在血清孵育前后，分别比较内皮细胞对SP修饰 的与未修饰的递送系统摄取量。
4.SP修饰的递送系统在正常小鼠体内跨BBB能力的评价，
正常小鼠(如昆明种，C57BL/6等)，尾静脉注射包载荧光素的递送系统，比较不同时间点小鼠脑内SP修饰与未修饰的递送系统的蓄积量。
5.SP修饰的递送系统在正常大鼠体内的药代动力学的评价
正常SD大鼠，尾静脉注射荧光素DiI标记的多肽修饰与未修饰的递送系统，在不同的时间点收集血液，分离血浆，通过荧光定量检测血浆中DiI的含量，以评价SP修饰的递送系统在大鼠体内的药代动力学。
6.SP修饰的递送系统在正常小鼠体内的免疫原性的评价
制备含Lipid A的多肽修饰与未修饰的递送系统，腹腔注射于Balb/c小鼠体内，四周内每七天注射一次，在每次注射后第七天眼眶取血，收集血浆冷冻备用。利用ELISA法检测不同时间点在小鼠体内产生针对PEG和SP的IgG和IgM含量，评价免疫原性。
7.SP修饰的递送系统体内抗肿瘤效果评价
通过荷原位U87神经胶质瘤模型裸鼠尾静脉注射SP修饰的递送系统、未修饰多肽的递送系统、游离药物和生理盐水，以模型裸鼠的中位生存期、肿瘤组织细胞凋亡和新生血管密度为指标评价SP修饰的递送系统体内抗肿瘤效果。
本发明经试验显示，所述的SP介导的药物或递药系统靶向LRP-1高表达的细胞和组织，具有跨生物膜屏障的能力，特别是跨血脑屏障(BBB)的能力，可用于脑肿瘤(含脑转移瘤和原发性脑肿瘤)及其它疾病的靶向诊断和治疗。
本发明实验显示SP修饰在递药系统表面，与血浆中的载脂蛋白结合，形成蛋白冠后增加血管内皮细胞的摄取；SP可同时介导药物跨血脑屏障，和/或靶向肿瘤新生血管和肿瘤细胞，其修饰的药物和递药系统在治疗脑肿瘤(含脑转移瘤和原发性脑肿瘤)及脑内其它疾病时可获得更好的治疗效果。
附图的简要说明
图1.SP修饰的脂质体与血浆蛋白的体外结合鉴定
SP通过化学偶联的方式修饰在脂质体表面得到SP-sLip,与小鼠血浆混合37℃孵育1h后，高速离心获得含有蛋白冠的脂质体沉淀。经Biorad 4％-20％梯度SDS-PAGE分离，快速银染法得到脂质体吸附的血浆蛋白成分分析(如附图1A所示)。结果发现，相比于甲氧基聚乙二醇修饰的长循环脂质体(sLip),SP-sLip吸附的血浆蛋白在45KDa(条带1)，38KDa(条带2)和25KDa(条带3)处的条带有显著性增加。将两者三处相同位置对应的PAGE胶经LC-MS/MS和western blot(如附图1B所示)结果分析，鉴定三处的蛋白分别是小鼠来源的ApoJ，ApoE和ApoA1。Western blot半定量法亦证实，SP-sLip相比于sLip吸附的三种载脂蛋白有显著增加(如附图1C-E所示)。
图2.荧光标记的sLip和SP-sLip在体内吸附血浆功能性载脂蛋白的鉴定
为了验证目标小肽修饰的脂质体在动物体内经全身给药后，仍能吸附相关的目的蛋白，我们将等量的DiI荧光标记的脂质体经尾静脉注射至小鼠体内，1h后取血，分离血浆中的含蛋白冠的脂质体。如附图2A所示，经过western blot方法检测到，相同含量的脂质体在小鼠体内吸附的三种载脂蛋白(ApoJ，ApoE，ApoA1)的含量有显著差异。这一结果证明，相对于普通脂质体，SP修饰的脂质体(SP-sLip)在小鼠体内能够快速并特异性的吸附血浆蛋白中的载脂蛋白。(见附图2B-D半定量分析)
图3.sLip和SP-sLip吸附血浆蛋白后，与LRP-1受体的结合活性评价
SP修饰的脂质体(SP-sLip)自身能够特异性吸附溶液中的重组蛋白LRP-1(如附图3A所示)。我们通过western blot方法证实SP-sLip与ApoE预孵育之后，仍保留与LRP-1结合的能力(如附图3B-C所示)。而附图3DE通过ELISA实验证明，游离的SP和修饰的脂质体(SP-sLip)均能够竞争固定在96孔板上SP与抗体相互作用，而血浆孵育之后的SP-sLip失去竞争抗体结合能力，说明脂质体表面的SP与ApoE等血浆蛋白结合之后，其功能域被封闭。SP-sLip表面特异性结合的载脂蛋白如ApoE具有与BBB受体LRP-1结合的活性，成为介导SP-sLip跨越BBB入脑的有效途径。
图4.血管内皮细胞对血浆孵育前后脂质体的摄取
SP修饰的脂质体(SP-sLip)与血浆共孵育形成蛋白冠后，内皮细胞对其摄取增加(如附图4A-B所示)。
图5.SP修饰的脂质体在小鼠体内的药动学参数和免疫原性评价
SP修饰在脂质体表面(SP-sLip)，未影响脂质体体内药动学参数(如附图5A所示)，也未增加普通长循环脂质体产生的免疫原性(以血液中IgG和IgM为评价指标，如附图5B-D)。图6.SP修饰的脂质体在小鼠体内入脑效率评价
通过体内检测小鼠脑内的荧光素，发现SP-sLip组跨BBB的量显著高于sLip组，说明SP可介导递送系统跨血脑屏障。
图7.载阿霉素的脂质体体内抗脑胶质瘤的药效
生理盐水组、DOX组、sLip/DOX组、SP-sLip/DOX组小鼠的中位生存期分别为：27天，31天，33天，50天，SP修饰的脂质体可显著延长脑原位瘤模型鼠的中位生存期。
图8.载阿霉素的脂质体对肿瘤组织中新生血管的影响
SP修饰的脂质体包载阿霉素组(SP-sLip/DOX)肿瘤组织内的血管密度显著低于未修饰组(sLip/DOX)，说明SP可介导递送系统靶向肿瘤新生血管。
图9.载阿霉素的脂质体对胶质瘤细胞凋亡的影响
SP修饰的脂质体包载阿霉素组(SP-sLip/DOX)肿瘤组织内细胞凋亡数量显著高于未修饰组，说明SP可介导递药系统靶向肿瘤细胞。
图10.安全性评价
SP在体外培养的神经细胞系PC12上并未显示出细胞毒性(如附图10A所示)，同时SP修饰的脂质体包载阿霉素组(SP-sLip/DOX)小鼠脏器的组织切片无明显异常(如附图 10B所示)，说明SP修饰在脂质体表面并未引起毒副作用。
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 制备脂质体
脂质体sLip/DiI和SP-sLip/DiI的制备：
脂质体sLip/DiI的制备：称取制备脂质体的膜材天然磷脂(HSPC)：7.85mg胆固醇:3.35mg；mPEG-2000-DSPE:2.78mg；DiI:0.4mg，溶于10mL CHCl 3中，40度水浴悬蒸成膜，真空干燥去除有机溶剂，将膜溶于1mL双蒸水中得60度水浴中摇匀水化，在脂质体挤压器经400nm，200nm，100nm孔径挤压得到sLip/DiI；
脂质体SP-sLip/DiI的制备：称取50mg Mal-PEG-DSPE溶于5mL CHCl 3中37度成膜，真空干燥半小时后溶于4mL双蒸水中，37度水化，超声以去除大颗粒。称取26mg SP-Cys蛋白溶于2mL双蒸水中，于上诉膜材料溶液混合，并用1mL双蒸水润洗容器后混合，加入40μL EDTA溶液(500mM，pH 8.0)，3mL PB溶液(0.1M，pH 7.4)，室温搅拌反应6h，使之无絮状沉淀。使用8000-10000Da孔径透析膜，蒸馏水中透析48h-72h，得到的溶液冻干制品为SP-PEG-DSPE；
称取制备脂质体的膜材HSPC：7.85mg胆固醇:3.35mg；mPEG-2000-DSPE:1.67mg；SP-PEG-DSPE:1.82mg；DiI:0.4mg，溶于10mL CHCl 3中，40度水浴悬蒸成膜，真空干燥去除有机溶剂，将膜溶于1mL双蒸水中得60度水浴中摇匀水化，在脂质体挤压器经400nm，200nm，100nm孔径挤压得到SP-sLip/DiI；
脂质体sLip/DOX和SP-sLip/DOX的制备：
脂质体sLip/DOX的制备：称取制备脂质体的膜材HSPC：7.85mg胆固醇:3.35mg；mPEG-2000-DSPE:2.78mg，溶于10mL CHCl 3中，40度水浴悬蒸成膜，真空干燥去除有机溶剂，将膜溶于1mL硫酸铵溶液(0.32M)中得60度水浴中摇匀水化，在脂质体挤压器经400nm，200nm，100nm孔径挤压得到硫酸铵溶液水化的sLip，经G50洗脱柱置换成生理盐水溶剂后，加入阿霉素水溶液混合(药脂比1:10)，经G50层析柱去除未包载阿霉素，得到sLip/DOX；
脂质体SP-sLip/DOX的制备：称取制备脂质体的膜材HSPC：7.85mg胆固醇:3.35mg； mPEG-2000-DSPE:1.67mg；SP-PEG-DSPE:1.82mg，其余步骤同上(脂质体sLip/DOX的制备)，得SP-sLip/DOX。
实施例2 脂质体在血清中形成蛋白冠后与LRP-1受体的结合活性及其被血管内皮细胞摄取的影响
脂质体在血清中形成蛋白冠的特征：
取C57BL/6小鼠的血清(含蛋白酶抑制剂，以EDTA为抗凝剂)与脂质体1:1混合，37℃孵育1h后，15000rpm离心30min，用冷的PBS洗2次后溶于30μL PBS中，以血清做阳性对照，PBS为阴性对照，加入6μL的SDS-PAGE上样缓冲液和3μL的β-巯基乙醇，煮沸10分钟使蛋白变性，用4-20％的聚丙烯酰胺胶使不同分子量的蛋白分离，快速银染试剂盒显色。将PAGE胶中的明显差异条带处(如红色箭头所示)及对照的同一位置处切下，分别用胰蛋白酶消化，重悬于0.1％的甲酸溶液中，LC-MS/MS分析各条带的蛋白组分，实验结果如图1所示；
体内实验实施方法如下：将荧光DiI标记的多肽修饰与未修饰的脂质体通过尾静脉注射到C57BL/6小鼠体内，1h后取血并低速离心分离血浆，蛋白冠分离方法如上述体外实验，通过SDS-PAGE和western blot方法鉴定其在体内吸附的蛋白冠成分，如图2所示。
血清孵育前后SP-sLip与LRP-1受体的结合活性：
ELISA法检测血清孵育前后SP-sLip与SP抗体的结合活性，以判断SP与受体的活性结合域在血浆蛋白相互作用后的变化，检测其吸附的载脂蛋白活性，具体操作如下：ELISA板中每孔加入0.1μg SP-PEG-DSPE，室温过夜，PBS洗3遍后，用3％BSA封闭1h，吸去BSA溶液，加入SP抗体37℃孵育1h，PBS洗三遍，加入预先与血清或者PBS孵育并梯度稀释的脂质体，在37℃孵育1h，PBS洗三遍，加入相应的辣根过氧化物酶标记的二抗，1h后与TMB显色液反应3-15min，用2M H 2SO 4终止反应，在450nm波长测其吸光度值，实验结果如图3所示。
SP-sLip在血清中形成蛋白冠后被血管内皮细胞摄取的影响：
血管内皮细胞bEND3的复苏：将冷冻bEND3细胞，快速融化并转移至预先加有培养液的离心管中，1000r/min离心3min后弃上清，加入含10％FBS的DMEM培养液，轻轻吹打均匀并滴加至培养皿中，混匀。于显微镜下观察细胞形态及生长情况并置于37℃、5％CO 2、相对湿度95％的培养箱中培养；
血管内皮细胞bEND3的培养：观察bEND3细胞的生长情况即细胞数量、形态和贴壁情况。吸弃旧培养液，加入新鲜的含10％FBS的DMEM培养液，混匀，在37℃、5％CO 2、饱和湿度的培养箱中继续培养，每天观察细胞生长情况，每隔2-3天传代，约10天左右细胞处于对数生长期，可用于体外细胞实验；
血管内皮细胞bEND3传代：吸弃培养液，用PBS冲洗2次，加入0.25％胰酶少许， 置培养箱中1min后加入2-3mL培养液终止消化，将该细胞悬液等份分装入数个离心管中，离心后弃去上清液，加入新的培养液后转入培养皿中，置培养箱中培养；
血管内皮细胞bEND3的冻存：细胞实验结束后，冻存细胞以便下次使用，预先配制冻存液(含10％DMSO和90％胎牛血清)，于4℃预冷。用胰酶消化细胞后加入预冷的冻存液，用滴管轻轻吹打混匀。在每支冻存管中加入1mL细胞液，密封后标记冻存细胞名称和冻存日期，置-80℃过夜，液氮罐中保存并登记备案；
将合适密度的bEND3接种于6孔板中，置于细胞培养箱中培养过夜，将荧光素DiI标记的脂质体(血清孵育的及未与血清孵育的)分别用不含血清的DMEM培养基稀释50倍，每孔加入1ml，与细胞共孵育4h，将培液吸出，快速用PBS洗两遍，胰酶消化细胞，含血清的培养基终止消化，离心，并用PBS洗一遍，最后细胞分散于300μL的PBS中，流式细胞仪测定摄取包载DiI脂质体的阳性细胞百分率和荧光强度(激发波长555nm，发射波长570nm)。实验结果如图4所示；
SP修饰对脂质体体内药动学参数及免疫原性的影响：
SD大鼠，每组3只，通过尾静脉注射sLip/DiI和SP-sLip/DiI，分别在给药前，5min，30min，1h，2h，4h，8h，12h和24h后，尾部取血(EDTA抗凝)，血样分离上清，并用正常大鼠血清梯度稀释血浆中的DiI，利用荧光分光光度计进行定量(激发波长555nm，发射波长570nm)，实验结果如图5a所示，腹腔注射载Lipid A的sLip和SP-sLip于Balb/c小鼠，每7天注射一次，每次注射后第七天取血备用，利用ELISA法检测不同时间点在小鼠体内产生针对PEG和SP的IgG和IgM含量，实验结果如图5b-c所示。
实施例3 SP修饰的脂质体可跨越BBB
体内实验证实荧光素DiI标记的SP修饰的脂质体(SP-sLip/DiI)可跨越血脑屏障：
将荧光标记的SP-sLip/DiI(磷脂含量10mg/mL,DiI含量0.4mg/mL)，尾静脉注射到C57BL/6小鼠体内(10μL/g)，1h后，用乙醚麻醉小鼠，取出脑组织，在4％的多聚甲醛中固定24h，30％蔗糖脱水，OCT包埋，冰冻切片，DAPI染细胞核，CD31抗体标记血管，置于显微镜下观察并拍照，荧光素DiI标记的甲氧基脂质体作对照，按相同步骤处理，实验结果如图6所示。
实施例4 载阿霉素的SP-sLip在体内的药效学实验
原位瘤模型鼠的中位生存期：
脑胶质瘤裸鼠模型的建立：取对数生长期的U87细胞，消化细胞并计数，用适量的PBS缓冲液悬浮，每只裸鼠接种6×10 5个细胞(分散于5μL PBS缓冲液中)，实验前将裸鼠用7％水合氯醛麻醉后，用脑立体定位仪固定，将悬浮细胞接种于纹状体部位(即前囟向前0.6mm，向右1.8mm，纵深3mm)，定期观察手术后裸鼠的状态；
将原位脑瘤模型裸鼠随机分成4组(n＝13)，分别在第7、9、11、13、15天记 录小鼠体重，尾静脉注射200μL生理盐水，阿霉素，sLip/DOX，SP-sLip/DOX，单次阿霉素的注射剂量为2mg/kg，记录模型裸鼠的生存时间，实验结果如图7所示。
载阿霉素的SP-sLip抑制肿瘤中新生血管和促肿瘤细胞凋亡实验：
原位脑胶质瘤模型鼠在给药完成后的第18天，水合氯醛麻醉，分离脑组织，多聚甲醛固定，脱水，石蜡包埋切片。通过CD31抗体的免疫荧光染色观察检测对新生血管的抑制效果，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，通过共聚焦荧光显微镜观察并拍照，实验结果如图8,9所示。
安全性评价：将梯度浓度的SP与神经细胞系PC12共培养，评价其在体外培养条件下的细胞毒性，同时将原位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，在显微镜下观察并拍照，实验结果如图10所示。
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。

Claims (15)

  1. 淀粉样蛋白β短肽在制备用于介导药物分子、荧光探针或递送系统靶向脑肿瘤和/或脑部其它疾病病灶的制剂中的应用，其中所述的淀粉样蛋白β短肽为特异性吸附血浆中载脂蛋白的淀粉样蛋白β短肽；优选地，所述脑肿瘤为脑转移瘤和/或原发性脑肿瘤。
  2. 根据权利要求1所述的用途，其特征在于，所述的淀粉样蛋白β短肽以共价键与影像物质X连接制得SP-X，用于脑肿瘤及脑内其它疾病病灶的示踪；
    优选地，所述的SP-X中，X是荧光分子Fluorescein或近红外染料分子Cy5、Cy5.5、Cy7、IR820、ICG、DiR、DiD、DiI；
    更优选地，所述脑肿瘤为脑转移瘤和/或原发性脑肿瘤。
  3. 根据权利要求1所述的用途，其特征在于，按所述的淀粉样蛋白β短肽以共价键与抗肿瘤药物Y连接制得SP-Y，用于脑肿瘤及脑内其它疾病病灶的靶向干预中；
    优选地，所述的SP-Y中，Y是吉非替尼、埃克替尼、安罗替尼、克唑替尼、厄洛替尼、奥希替尼、阿雷替尼、紫杉醇、多西他赛、卡巴他赛、阿霉素、表阿霉素、喜树碱、羟基喜树碱、9-硝基喜树碱或长春新碱小分子抗肿瘤药物；和/或
    所述的SP-Y中，Y是p53激活肽或多肽毒素多肽抗肿瘤药物；
    更优选地，所述脑肿瘤为脑转移瘤和/或原发性脑肿瘤。
  4. 根据权利要求1所述的用途，其特征在于，所述的淀粉样蛋白β短肽用共价键与聚乙二醇-Z连接制得的SP-聚乙二醇-Z，用于制备纳米递送系统；
    优选地，所述的SP-聚乙二醇-Z中，Z是磷脂、聚乳酸、乳酸羟基乙酸共聚物或聚己内酯；和/或
    优选地，所述的纳米递送系统选自以下一种或多种：脂质体递送系统、胶束递送系统、纳米圆盘递送系统和和聚合物纳米粒递送系统；
    更优选地，所述的SP-聚乙二醇-磷脂用于制备脂质体递送系统、胶束递送系统或纳米圆盘递送系统；和/或所述的SP-聚乙二醇-聚乳酸、SP-聚乙二醇-乳酸羟基乙酸共聚物、SP-聚乙二醇-聚己内酯，用于制备胶束递送系统和聚合物纳米粒递送系统；
    进一步优选地，所述的脂质体递送系统、胶束递送系统、纳米圆盘递送系统或聚合物纳米粒递送系统，用于包载诊断分子，进行脑肿瘤或脑部其它疾病病灶的示踪；优选地，所述的递送系统所包载诊断分子是5-羧基荧光素5-FAM或近红外染料Cy5、Cy5.5、Cy7、IR820、ICG、DiR、DiD、DiI；和/或
    所述的纳米粒递送系统、脂质体递送系统、胶束递送系统、聚合物纳米粒递送系统或纳米圆盘递送系统，用于包载抗肿瘤药物，进行脑肿瘤或脑部其它疾病病灶的靶向干预；优选地，所述的递药系统所包载药物是吉非替尼、埃克替尼、安罗替尼、克唑替尼、厄洛替尼、奥希替尼、阿雷替尼、紫杉醇、多西他赛、卡巴他赛、阿霉素、表阿霉素、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、p53激活肽或多肽毒素；
    更进一步优选地，所述脑肿瘤为脑转移瘤和/或原发性脑肿瘤。
  5. 一种用于制备介导药物分子、荧光探针或递送系统靶向脑肿瘤或脑部其它疾病病灶制剂的淀粉样蛋白β短肽，其特征在于，所述的淀粉样蛋白β短肽为特异性吸附血浆中载脂蛋白的淀粉样蛋白β短肽；优选地，所述脑肿瘤为脑转移瘤和/或原发性脑肿瘤。
  6. 一种用于脑肿瘤及脑内其它疾病病灶的示踪分子，其特征在于，所述示踪分子为权利要求5所述的淀粉样蛋白β短肽以共价键与影像物质X连接制得SP-X；
    优选地，所述的SP-X中，X是荧光分子Fluorescein或近红外染料分子Cy5、Cy5.5、Cy7、IR820、ICG、DiR、DiD、DiI；
    更优选地，所述脑肿瘤为脑转移瘤和/或原发性脑肿瘤。
  7. 一种用于脑肿瘤及脑内其它疾病病灶的靶向干预的药物，其特征在于，所述药物为权利要求5所述的淀粉样蛋白β短肽以共价键与抗肿瘤药物Y连接制得SP-Y；
    优选地，所述的SP-Y中，Y是吉非替尼、埃克替尼、安罗替尼、克唑替尼、厄洛替尼、奥希替尼、阿雷替尼、紫杉醇、多西他赛、卡巴他赛、阿霉素、表阿霉素、喜树碱、羟基喜树碱、9-硝基喜树碱或长春新碱小分子抗肿瘤药物；和/或
    所述的SP-Y中，Y是p53激活肽或多肽毒素多肽抗肿瘤药物；
    更优选地，所述脑肿瘤为脑转移瘤和/或原发性脑肿瘤。
  8. 一种纳米递送系统，其特征在于，所述纳米递送系统包含权利要求5所述的淀粉样蛋白β短肽用共价键与聚乙二醇-Z连接制得的SP-聚乙二醇-Z；
    优选地，所述的SP-聚乙二醇-Z中，Z是磷脂、聚乳酸、乳酸羟基乙酸共聚物或聚己内酯；
    更优选地，所述的纳米递送系统选自以下一种或多种：脂质体递送系统、胶束递送系统、纳米圆盘递送系统和和聚合物纳米粒递送系统；
    进一步优选地，当所述的SP-聚乙二醇-Z为SP-聚乙二醇-磷脂时，所述的纳米递送系统选自以下一种或多种：脂质体递送系统、胶束递送系统和纳米圆盘递送系统；当所述的SP- 聚乙二醇-Z为SP-聚乙二醇-聚乳酸、SP-聚乙二醇-乳酸羟基乙酸共聚物、SP-聚乙二醇-聚己内酯时，所述的纳米递送系统为胶束递送系统和/或聚合物纳米粒递送系统；
    进一步优选地，所述的脂质体递送系统、胶束递送系统、纳米圆盘递送系统或聚合物纳米粒递送系统，用于包载诊断分子，进行脑肿瘤或脑部其它疾病病灶的示踪；优选地，所述的递送系统所包载诊断分子是5-羧基荧光素5-FAM或近红外染料Cy5、Cy5.5、Cy7、IR820、ICG、DiR、DiD、DiI；和/或
    进一步优选地，所述的纳米粒递送系统、脂质体递送系统、胶束递送系统、聚合物纳米粒递送系统或纳米圆盘递送系统，用于包载抗肿瘤药物，进行脑肿瘤或脑部其它疾病病灶的靶向干预；优选地，所述的递药系统所包载药物是吉非替尼、埃克替尼、安罗替尼、克唑替尼、厄洛替尼、奥希替尼、阿雷替尼、紫杉醇、多西他赛、卡巴他赛、阿霉素、表阿霉素、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、p53激活肽或多肽毒素；
    更进一步优选地，所述脑肿瘤为脑转移瘤和/或原发性脑肿瘤。
  9. 根据权利要求8所述的纳米递送系统的制备方法，其特征在于，所述方法包括以下步骤：
    称取天然磷脂，胆固醇，甲氧基-PEG-DSPE，与SP-聚乙二醇-Z，模型药物或探针分子，溶解于溶媒中，成膜，水化，过膜，柱层析除去游离探针分子或药物，制得所述纳米递送系统；和/或
    称取天然磷脂，胆固醇，甲氧基-PEG-DSPE，与SP-聚乙二醇-Z溶解于溶媒中，成膜，水化，过膜，主动包封探针分子或药物，柱层析除去游离探针分子或药物，制得所述纳米递送系统。
  10. 一种药物组合物，其特征在于，所述药物包含：1)权利要求5所述的淀粉样蛋白β短肽、权利要求6所述的示踪分子、权利要求7所述的药物和/或权利要求8所述的纳米递送系统；以及2)药学上可接受的载体。
  11. 一种诊断制剂，其特征在于，所述诊断制剂包含：权利要求5所述的淀粉样蛋白β短肽、权利要求6所述的示踪分子、权利要求7所述的药物和/或权利要求8所述的纳米递送系统。
  12. 一种用于治疗脑部疾病的方法，其特征在于，所述方法包括：对有需要的受试者给予权利要求5所述的淀粉样蛋白β短肽、权利要求6所述的示踪分子、权利要求7所述的药物和/或权利要求8所述的纳米递送系统；
    优选地，所述脑部疾病为脑肿瘤或脑部其它疾病；
    更优选地，所述脑肿瘤为脑转移瘤和/或原发性脑肿瘤。
  13. 一种用于诊断脑肿瘤及脑内其它疾病病灶的方法，其特征在于，所述方法包括：对有需要的受试者给予权利要求5所述的淀粉样蛋白β短肽、权利要求6所述的示踪分子、权利要求7所述的药物和/或权利要求8所述的纳米递送系统；优选地，所述脑肿瘤为脑转移瘤和/或原发性脑肿瘤。
  14. 一种脑肿瘤或脑部其它疾病病灶的靶向干预方法，其特征在于，所述方法包括：对有需要的受试者给予权利要求5所述的淀粉样蛋白β短肽、权利要求6所述的示踪分子、权利要求7所述的药物和/或权利要求8所述的纳米递送系统；优选地，所述脑肿瘤为脑转移瘤和/或原发性脑肿瘤。
  15. 一种药物介导方法，其特征在于，所述方法包括：对有需要的受试者给予权利要求5所述的淀粉样蛋白β短肽、权利要求6所述的示踪分子、权利要求7所述的药物和/或权利要求8所述的纳米递送系统。
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